Re: [Histonet] Fast Green / Sirius Red - Unknown blue features

[Liz Chlipala via Histonet]

David

We have found that mordanting in bouins (just like you would a Masson's 
trichrome) prior to completing a standard Picrosirius Red stain protocol that 
we get very consistent results.  I think this method has been published but I 
do not have access to the reference currently.  Its worth a shot for your PSR 
stain.  What I do know is that the brief rinse in acetic acid post the PSR 
stain is necessary, that seems to remove any excess red/pink that may stain the 
cytoplasm of the cells, not much of a distractor for manual reads for fibrosis 
but may be problematic if you are utilizing an image analysis solution.  I 
can't comment on the other method.  If you like I can forward on our PSR method 
in a separate email.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com
 
Take a look at our most recent publications:



https://journals.lww.com/appliedimmunohist/Abstract/publishahead/An_Image_Analysis_Solution_For_Quantification_and.98711.aspx

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879147/

-Original Message-
From: David Burk via Histonet  
Sent: Thursday, June 22, 2023 6:38 AM
To: John Kiernan ; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Fast Green / Sirius Red - Unknown blue features

Dr. Kiernan,

You are correct regarding the source of the technique we are emulating. We have 
had issues with consistency in keeping the yellow picric acid color in 
cytoplasm when performing the traditional PSR stain possibly due to variability 
in what each individual considers 'quick' in their rinses and ethanol steps. 
Looking in the literature, you can find many publications that show PRS-stained 
tissue with golden-yellow / straw-colored cytoplasm (ideal), pale pink, or even 
virtually 'clear'.

The stain we are currently using is from a bottle likely purchased in the 80's 
or 90's and does bear the label you describe. Allied Chemical in Morristown, NJ 
which no longer exists. I'll try a newer bottle we have and see if the results 
remain the same. Our Direct Red 80 is from Sigma-Aldrich (365548) and does not 
bear the certification mark you mention. However, as we do see the expected 
collagen staining in tissues (and the stained material exhibits birefringence), 
I am fairly confident that it is, at the very least, 'OK'. If you happen to 
know of vendors that routinely offer certified dyes, I'd be happy to utilize 
them in the future.

As you mention in your separate email, what I may consider blue may appear 
differently to others either by technology or anatomy/physiology. Regardless, 
they are unexpected and have not been described in the literature and, in that 
regard, present a potential source of confusion.

Best,
David




From: John Kiernan 
Sent: Wednesday, June 21, 2023 11:36 PM
To: histonet@lists.utsouthwestern.edu ; 
David Burk 
Subject: Re: Fast Green / Sirius Red - Unknown blue features

Your technique is the one first (I think) published by Lopez-De Leon A & 
Rojkind M (1985) A simple micromethod for collagen and total protein 
determination in formalin-fixed paraffin-embedded sections. J. Histochem. 
Cytochem. 33: 737-743. The photos in that paper show some of the collagen 
almost black - surely taking up both red and green dyes. More recent papers 
describe exactly the same method, and there are also some variants. Your 
technique, with an acid rinse after staining for an hour, then quick transition 
to rapid dehydration in 100% alcohol, is essential for any valid picro-sirius 
staining.

According to the entry for fast green FCF (CI 42053) in Conn's Biological 
Stains (10th ed, p.180-182), "chemically distinct blue-green dyes have been 
supplied under this name". Are you sure your fast green FCF is the real McCoy? 
Is it from a batch certified by the Biological Stain Commission? The jar of dye 
powder should have a small bluish label, with features that make forgery 
difficult. See 
https://us-west-2.protection.sophos.com?d=biologicalstaincommission.org=aHR0cHM6Ly9iaW9sb2dpY2Fsc3RhaW5jb21taXNzaW9uLm9yZy9jZXJ0aWZpZWQtYmlvbG9naWNhbC1keWVzc3RhaW5zLw===NWYzYTk2OTEyMDVmMDkwZWJiNmJlNzc2=WjVWeGtKVHl5R3FneU9tQjkxK1gydnlWY2Y4Yk9ESGZ1SjBkY1c3ZmxRST0==279d4c37c7764be8a8cd92a53a018013=AVNPUEhUT0NFTkNSWVBUSVZpNWvhfkvsy0HBXeMJqMJXLVUzTeOGssiQ7jAnNTZ6aDtn6HKp77NuWaELZg1b57k
 for pictures and other information. There are companies selling "certified 
stains" that have not been tested and certified by the Biological Stain 
Commission. Caveat emptor!

The Biological Stain Commission is a not-for-profit corporation that has been 
providing third-party quality control and other services for vendors and users 
of stains for 100 years.

Just a few thoughts; I could add more, but probably this letter already is too 
long for the Histonet censors.

John Kiernan
Professor Emeritus, Anatomy & 

Re: [Histonet] Desperately seeking a certain IHC expert

[Liz Chlipala via Histonet]

Joe Myers biocate

Sent from my Windows 10 device

From: Maria Cruz via Histonet
Sent: Monday, February 3, 2020 7:03 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Desperately seeking a certain IHC expert

Hey everybody - This is an unusual request or at least I think it is. I’m
hoping that someone can help me find and connect with a guy that I heard
speak at the national histo-meeting. I’ve also seen his name as a
presenter at several state meetings to. Hes involved with the national
society and seems to always give presentations on IHC topics. I’m pretty
sure that He works for one of the major IHC companies. The lab I work in
has major IHC issues and he always seems to be able to answer everyone’s
questions. Thanks in advance.
Maria
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Re: [Histonet] IHC on Pig Tissues

[Liz Chlipala via Histonet]

Lori

You can't use a dual label detection system.  We have found most mouse polymers 
will cross react with porcine tissue.  We know that the dako one does.  You 
need to use the rabbit envision for rabbit monoclonal antibodies or with mouse 
monoclonals you need to use a rabbit anti-mouse and then a rabbit envision 
detection system.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Lori Sorrells via Histonet 
Sent: Wednesday, January 29, 2020 1:35 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC on Pig Tissues

Good afternoon-
Has anyone run into problems when trying to perform IHC using rabbit mono or 
polyclonal antibodies on pig tissues? I typically do not have any issues with 
nonspecific staining or high background when using mouse monoclonal antibodies. 
I have used a variety of blockers including various sera in the diluents and 
blocking reagents. All samples are FFPE. I routinely use the DAKO EnVision+ 
Dual HRP system. Thanks in advance!

Lori Sorrells
Research Associate II
Revivicor, Inc.
1700 Kraft Drive, Suite 2400
Blacksburg, VA 24060

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Re: [Histonet] H trouble shooting

[Liz Chlipala via Histonet]

That an image of incomplete dehydration the samples have not been processed 
properly and adequately dehydrated.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Wang, Weixi via Histonet 
Sent: Wednesday, January 15, 2020 11:20 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] H trouble shooting

Hi,
I have some issues on the staining quality of biopsies. Please see the image 
link below, the overall staining looks good but the edge looks weird, no 
nuclear staining picked up. Under-fixed? Over-dehydrated in the processor? Or 
dried during grossing?
You expertise is greatly appreciated!

https://imgur.com/gallery/kpLqRT9


Thank you,
Weixi




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Re: [Histonet] guidelines for using a microwave designated for food, but used to heat up histogel

[Liz Chlipala via Histonet]

You don’t need to heat up histogel in a microwave its quicker that way but if 
you set an oven at around 65 to 70 it will melt in around 20 minutes or so, 
that’s what we do.  It can get messy in a microwave.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Eileen Akemi Allison via Histonet 
Sent: Thursday, December 12, 2019 6:32 AM
To: Kathleen S Cormier 
Cc: Histonet 
Subject: Re: [Histonet] guidelines for using a microwave designated for food, 
but used to heat up histogel

Went on line to get MSDS regarding HistoGel. Here’s the link. 
https://www.medline.com/media/catalog/Docs/MSDS/MSD_SDSD91968.pdf 


Happy eating from a microwave which has been used at MD Anderson for HistGel!

Best regards,
Akemi

> On Dec 12, 2019, at 5:18 AM, Kathleen S Cormier 
>  wrote:
>
> Can I share that we had a pathologist (back in the day) use our lab microwave 
> to re-heat his coffee? It had Schiff’s reagent stains all over the inside of 
> it. Some people really don’t have any common sense…
>
>
> Kathy Cormier
> Core Leader
> Hope Babette Tang Histology Facility
> Koch Institute for Integrative Cancer Research at MIT
> 500 Main Street, 76-182
> Cambridge, MA 02139
> 617-258-8183
> corm...@mit.edu 
> 
> https://ki.mit.edu/sbc/histology 
> 
>
>
>
>
>
>
>
>
>
>> On Dec 12, 2019, at 7:12 AM, Eileen Akemi Allison via Histonet 
>> mailto:histonet@lists.utsouthwestern.edu> 
>> >
>>  wrote:
>>
>> Good morning histopeeps:
>>
>> We recently brought on an Ophthalmology Pathologist from MD Anderson and 
>> they use histogel for orienting their thin eye specimens and need a 
>> microwave to heat it up. We do not have a microwave in the lab for their use 
>> but I had ordered one for them. I just found out they used the microwave in 
>> our lab lounge to heat up histonet which was opened and being stored in our 
>> gross area refrigerator.
>>
>> Do any of you know the CAP regulation against use of laboratory reagents 
>> being used in a microwave being used for food consumption? I ended up 
>> removing the microwave they used and put it in the histology lab. Our 
>> Director wants me to write a formal letter to submit to him.
>>
>> Thank you in advance for your impute!
>>
>> Akemi Allison, BS, HT/HTL (ASCP)
>>
>>
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>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>

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Re: [Histonet] Roche Symphony and Ultra users

[Liz Chlipala via Histonet]

It might be the coverglass we have seen bubbles in the glass itself, it looks 
like there is water on the slide, but it's actually bubbles in the coverglass

Sent from my Windows 10 device

From: Victoria Baker via Histonet
Sent: Friday, August 30, 2019 10:44 AM
To: Histo Net list server
Subject: [Histonet] Roche Symphony and Ultra users

Happy Friday!

I'm looking to see if anyone out there is using these two specific
instruments together.

For about 8 or 9 months we have been experiencing what looks like water or
water droplets on the IHC slides. It's not on all slides and we can go a
span of time with no issues only to have it come back. It's not antibody
specific either. We wash (Dawn dish detergent) and then load into the
Symphony.

I know there is the human factor, but right now I am just looking to see if
anyone is or has experienced this.

Thanks and enjoy your weekend.

Vikki
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Re: [Histonet] Antibody Validation CLIA

[Liz Chlipala via Histonet]

Keep in mind like Lacy said its cases and not slides so you could place 
multiple cases on one slide or create a simple array using disposable biopsy 
punches.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
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From: Normington Lacy via Histonet 
Sent: Friday, March 16, 2018 11:17 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Antibody Validation CLIA


CAP suggests running 10 negative and 10 positive cases for non-prognostic 
markers.
CAP required running 20 negative and 20 positive cases for prognostic markers.

In the event the case volume is less than the suggested 10 and 10 cases for 
non-prognostic markers, the reason for that decision should be stated in the 
validation. Ultimately, the decision is up to your director.


Lacy Normington, HTL(ASCP)CM
Manager, Surgical Pathology Lab Services UW Health
600 Highland Avenue
Madison, WI 53792-2472



-Original Message-
From: Paula via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Friday, March 16, 2018 8:55 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Antibody Validation CLIA

WARNING: This email appears to have originated outside of the UW Health email 
system.
DO NOT CLICK on links or attachments unless you recognize the sender and know 
the content is safe.




Hello,



We've been discussing about the quantity of slides to run as a validation for 
IHC antibodies. We are governed by CLIA, and we would like to know if there is 
a set number of slides to run for a particular antibody we would like to bring 
in-house for Validation. I think CAP requires 20 slides..?
And so we are asking if there is a requirement with CLIA to run a certain 
number of slides, or is it up to us (the laboratory director) to decide how 
many slides to run for Validation/Verification.



Thank you in advance

Paula

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Re: [Histonet] Image analysis on breast markers

[Liz Chlipala via Histonet]

Teresa

CAP should be releasing a paper soon, within the next couple months.  
Quantitative Image Analysis (QIA) for HER2 Immunohistochemistry for Breast 
Cancer, it will address the question that  you have.  Keep any eye out for it.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Lima, Teresa via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Thursday, January 25, 2018 12:48 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Image analysis on breast markers

Hello all,
I am wondering if any of you out there that do image analysis of breast markers 
have a technologist select the field of view to be analyzed by the system. Our 
techs do this with no supervision by a pathologist. In many instances the 
pathologist changes the analyzed result without logging in to the system to 
check which areas were selected by the tech. This makes me just a bit nervous 
and I will be interested to see what you all are doing. Thanks and I look 
forward to your replies.

Many thanks,
Terri

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Re: [Histonet] Mouse bladder

[Liz Chlipala via Histonet]

I would try histogel instead of agar.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

From: Heather Marlatt via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, December 19, 2017 9:12 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Mouse bladder

Does anyone have a good protocol for mouse bladder embedded in agar that
they are willing to share? We have mouse bladders in 2% agar and have tried
a few different protocol variations from our usual mouse but the agar keeps
shriveling up.

Thanks
Heather
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Re: [Histonet] Beaker with or without Vantage

[Liz Chlipala via Histonet]

I'm a member of API - Association for Pathology Informatics  and they have a 
list server similar to the histonet and there is always posts about Epic 
Beaker.  You might find some information on their website.

https://www.pathologyinformatics.org/

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
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From: Morken, Timothy via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, November 28, 2017 9:48 AM
To: Histonet 
Subject: Re: [Histonet] Beaker with or without Vantage

I am interested as well. "They" are threatening us with a move to Beaker in the 
future


Tim Morken
Pathology Site Manager, Parnassus
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center

-Original Message-
From: Blake Taylor via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, November 28, 2017 8:10 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Beaker with or without Vantage

I'm looking for anyone out there that has switched to Beaker for AP, Also do 
you use Ventana Connect with Beaker? Did you choose to use the Beaker tracking 
system or is anyone using Beaker in conjunction with Vantage? Our Hospital is 
in the beginning phase of moving from Copath Sunquest to Beaker (2017 version) 
. Any thoughts of what has gone well and what has not would be appreciated.

Thanks so much

Blake Taylor
Surgical Pathology Supervisor
Lexington Medical Center
803-936-8214
bcdu...@lexhealth.org

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Re: [Histonet] Tracking lot numbers

[Liz Chlipala via Histonet]

Teri

When we change the tissue processor or H stainer instead of just recording a 
"check mark" that the solution has been changed we record the lot numbers on 
the form instead.  We also have a from that is posted above the tissue 
processor that we fill in with dry erase the current lot numbers and expiration 
dates of the solutions.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
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From: Johnson, Teri via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Tuesday, October 24, 2017 3:54 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Tracking lot numbers

Hi Histonetters,

I need to find out what labs are doing for best practices to track reagent lot 
numbers in their tissue processor as they rotate reagents. We currently have a 
preparation log where we track the lot numbers, but by the time we do a 
rotation we may be using a different lot.

As always, thanks for your help and input.


Teri Johnson, HT(ASCP)QIHC
Manager, Clinical Trial Testing

T +1 760 516 5954
teri.john...@navigatebp.com

Navigate BioPharma, Inc.
A Novartis Subsidiary
1890 Rutherford Rd.
Carlsbad, CA  92008
USA



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Re: [Histonet] Old control blocks

[Liz Chlipala via Histonet]

Elaine

We have the temperature of our cryostat calibrated yearly by an outside 
calibration company.  If the thermometer you are using inside the cryostat has 
been calibrated and is traceable  I think you might have an issue with the 
temperature reading that is being displayed on your cryostat.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
l...@premierlab.com
www.premierlab.com

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
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From: Elaine allison Hoffman via Histonet 
[mailto:histonet@lists.utsouthwestern.edu]
Sent: Thursday, October 5, 2017 9:42 AM
To: Histonetters Histonet 
Subject: [Histonet] Old control blocks

Hi everyone,
Just want to put a question out there. . . .
How long can we keep control blocks (paraffin embedded tissue) before they go 
bad?  I just went through our old control blocks in our filing container and 
some of our control blocks have dates as far back as 10 years ago.  They have 
not been refrigerated or anything but didn't know if they should be thrown out 
and start over or how long they are good for?  They are filed in alphabetical 
order, anything from amyloid controls to yeast control blocks and many others 
in-between.  Or probably should be tested to see just how positive the staining 
results turn out to be.  I was just wondering if anyone knew off the top of 
their head, lol.
Another brain teaser
Our cryostat is displaying a temperature of -23 degrees but the thermometer 
sitting inside the chamber is reading -15 degrees.  The thermometer was just 
recently purchased and does not need calibrated or anything.  Our supervisor 
thought we should have a thermometer inside the cryo-chamber just to be certain 
that the digital display is accurate.  Now we have a dilemma!  Did anyone else 
experience this problem with the temperature readings with their cryostat?  Not 
really sure what to do about the different readings.  What should the correct 
temperature be anyways?
We would really appreciate any feed-back or suggestions.
Thank you,

Elaine Hoffman, HT(ASCP)
Steward Trumbull Memorial HospitalWarren, OH
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RE: [Histonet] Paraffin processing and embeding of Chondrocyte pellets.

[Liz Chlipala]

Bret

We have done a bit of work with these types of samples, the first thing
we do is remove the formalin and add eosin to the container for about 5
minutes.  Then we place the pellet (using a plastic disposable pipettor)
in a mesh bag and process on our tissue processor on a cycle that is set
at 7 minutes per station, we also used propar instead of xylene (3
changes).  We used to process by hand but this method works just as
well.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Clough,
Bret
Sent: Tuesday, June 14, 2011 8:52 PM
To: Histonet list serv. (histonet@lists.utsouthwestern.edu)
Subject: [Histonet] Paraffin processing and embeding of Chondrocyte
pellets.

Hello Everyone,
 I was wondering if anyone has a protocol for paraffin embedding
chondrocyte pellets that they would be willing to share with me? Any
help would be greatly appreciated.

Thanks,
 Bret

Bret Clough
TAMHSC

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RE: [Histonet] (no subject)

[Liz Chlipala]

Dorothy

I would assume that you would just run the validation as you would any
other antibody that is not a pre dilute.  Run the antibody protocol on
the number of tissues that you normally do to validate, evaluate and
record the results.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dorothy
Glass
Sent: Wednesday, June 15, 2011 9:41 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] (no subject)

Dear Histonets, 
How is it possible to validate a antibody on the Ventana Ultra or XT,
when the 
antibodys afre prediluted or made into a prep-kit?
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[Histonet] RE: [IHCRG] laminin and collagen4 in cat and dog

[Liz Chlipala]

Margaret

 

We have used the Collagen IV listed below on canine but not feline, for
laminin we have not used the antibody from abcam on canine or feline but
the spec sheet states it works on most mammals.

 

Good Luck

 

Liz

 

Collagen IV

SouternBio

0340-01

UNLB

Goat

 

Laminin

abcam

ab11575

 

Rabbit

 

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

-Original Message-
From: ih...@googlegroups.com [mailto:ih...@googlegroups.com] On Behalf
Of Perry, Margaret
Sent: Thursday, June 02, 2011 12:05 PM
To: ih...@googlegroups.com; histonet@lists.utsouthwestern.edu
Subject: [IHCRG] laminin and collagen4 in cat and dog

 

We want to use laminin  and Collagen IV to sort out the origin of some
spindle cell tumors.  Specifically, malignant peripheral nerve sheath
tumors reportedly show laminin and collagen IV basement membranes vs.
Fibrosarcoma which does not.  We need antibodies that will work in both
cat and dog.  Is there anyone who has had success with these? Do you
have any recommendations for these antibodies?   

 

 

Margaret Perry HT(ASCP) 

Dept of Veterinary and  Biomedical services

Box 2175

South Dakota State University

Brookings SD 57007

605-688-5638

 

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RE: [Histonet] Fume hood

[Liz Chlipala]

We have a service come out and inspect our hoods once a year.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amber
McKenzie
Sent: Wednesday, June 01, 2011 2:32 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Fume hood 

What are you using to check your fume hood's with for QC purposes?


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RE: [Histonet] IHC pos. neg. control question

[Liz Chlipala]

Amos

Isotype negative controls are based upon protein concentration not
dilution.  They must be the same protein concentration of the primary
antibody at the dilution you are using.  Using the same dilution will
not work unless both stock solutions (primary antibody and isotype
control) are at the same protein concentration which is rarely the case.

Liz 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos
Brooks
Sent: Friday, May 20, 2011 2:16 PM
To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC pos.  neg. control question

Hi,
 This is simply a question of definitions. They are actually both
right.
If you have the patient tissue as a negative control you are not using
the
primary antibody on it but are replacing it (ideally) with an Ig with
the
same isotype AND dilution (universal negative is stupid). So if your
primary
is an IgG1 from a mouse and you use it at 1:100, your negative control
would
be a non-immune mouse serum IgG1 diluted to 1:100. Running this on
unrelated
material will not show you anything in this case. what it will show you
is
that the detection system you are using is specific to the antibody you
put
on the tissue and not to A) something else in the tissue or B) something
on
the Ig that is not the epitope itself.
 Although, If you run the same primary antibody on tissue that you
expect will not react with the primary antibody, this would prove that
the
antibody reaction is specific to the antigenin question. This does raise
the
question of what to do about ubiquitous antigens (like ubiquitin) that
are
actually everywhere. Every cell has a cell cycle so they will all at one
point or another show proliferation or degeneration for example. Does
this
mean you don't run a negative control? No you need to look at it in the
perspective of expected results.
 There are various ways of using negative (and positive) controls.
It
would actually be cost prohibitive to run ALL the possible positive and
negative controls for every test that we as histologists do. We need to
discuss with our pathologists what they want to have done to give them
the
confidence in our testing to support their diagnosis. But remember that
just
because another lab and another pathologist does it differently doesn't
mean
it is wrong. It's just different.

Happy Friday Folks,
Amos


Message: 5
Date: Thu, 19 May 2011 13:25:49 -0400
From: Jean-Martin Lapointe jm.lapoi...@accellab.com
Subject: [Histonet] IHC pos.  neg. control question
To: histonet@lists.utsouthwestern.edu
Message-ID:
   
befd613bd39142499989f836556ddc8301272...@ace.accellab.lan
Content-Type: text/plain;   charset=iso-8859-1

Hi Curt,
I agree with your pathologist. The section that you use as a negative
control (without primary) in an IHC run should ideally be tissue that is
a
known positive, and of the same nature as the test sample. Therefore the
best sample is often a section of your positive control tissue.
The purpose of this negative control is to make sure that any positive
staining observed in the test sample is not due to a spurious
cross-reaction, unrelated to the primary. I don't think the issue per se
is
that you are using tissue from the same patient; rather, it is that you
are
using a sample for which the staining characteristics are not known. For
instance, if are using a lymph node section as a negative, for a stain
that
targets an epithelial marker (eg Her2) in the test breast sample, then
your
negative tissue is not appropriate, because since lymph node contains no
epithelial tissue, it will not stain no matter what. Therefore if your
test
sample shows a positive reaction in the epithelial tissue, but for some
reason that reaction is a spurious false-positive, then the lymph node
negative will not reveal that.
I realize that this is all very theoretical and hypothetical, but I
understand the pathologist wanting to be confident in the knowledge that
all
potential technical issues are eliminated before making his diagnosis.

Jean-Martin
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RE: [Histonet] IHC pos. neg. control question

[Liz Chlipala]

No problem Amos, I have a one page handout that I use for workshops and
presentations on how to do the math on this, it can be tricky, so if
anyone is interested just e-mail me.  Since we are talking about matched
isotype negative controls, essentially three things need to be taken
into consideration:

 

1.  The isotype of the primary antibody
2.  The protein concentration of the working dilution of the primary
antibody
3.  The antibody form - (affinity purified, culture supernatant,
ascities fluid, etc.) the isotype negative control needs to match the
primary antibody - some spec sheets list appropriate isotype negative
control reagents some do not

 

There is one caveat that I would like to address for those that are
working with animal tissues - you need to check the spec sheet on your
negative control reagents to make sure that it does not cross react with
the species you are working with.  If it does then you may get staining
in your negative control slide.

 

Here is a table that was generated from one of the older versions of the
dako handbook that addresses the antibody form and suggested negative
control reagents.

 

Primary Antibody Type

Suggested Negative Control Reagent

Monoclonal Mouse - produced in ascites

Antibody produced in ascites, same isotype as the primary antibody  OR

Normal nonimmune mouse serum

Monoclonal Mouse -

produced in tissue culture

Antibody produced in tissue culture, same isotype as the primary
antibody

Polyclonal Rabbit or Goat - immunoglobulin fraction

Normal rabbit or goat immunoglobulin fraction

Solid-phase absorbed Polyclonal Rabbit - immunoglobulin fraction

Rabbit immunoglobulin fraction - solid phase absorbed

Polyclonal Rabbit - whole serum 

Normal or nonimmune rabbit serum, whole serum

 

Hope this helps, and everyone have a great weekend

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

-Original Message-
From: Amos Brooks [mailto:amosbro...@gmail.com] 
Sent: Friday, May 20, 2011 3:05 PM
To: Liz Chlipala
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] IHC pos.  neg. control question

 

Thanks Liz,
 You are absolutly right there, but have you ever noticed some folks
eyes glaze over when you say that they must both be run at the same
ug/mL? I would be lying if I said it never happened to me until I forced
myself to work it out. Once you do though it isn't too bad.
Unfortunately people don't think about this much. They want to put a
slide on a machine and walk away without thinking about what they are
doing. I guess I was oversimplifying the description. Thanks for
pointing that out as it is actually an important aspect to the
dilutions.

Amos

On Fri, May 20, 2011 at 4:25 PM, Liz Chlipala l...@premierlab.com
wrote:

Amos

Isotype negative controls are based upon protein concentration not
dilution.  They must be the same protein concentration of the primary
antibody at the dilution you are using.  Using the same dilution will
not work unless both stock solutions (primary antibody and isotype
control) are at the same protein concentration which is rarely the case.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 tel:%28303%29%20682-3949 
fax (303) 682-9060 tel:%28303%29%20682-9060 
www.premierlab.com


Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos
Brooks
Sent: Friday, May 20, 2011 2:16 PM
To: jm.lapoi...@accellab.com; histonet@lists.utsouthwestern.edu

Subject: [Histonet] IHC pos.  neg. control question

Hi,
This is simply a question of definitions. They are actually both
right.
If you have the patient tissue as a negative control you are not using
the
primary antibody on it but are replacing it (ideally) with an Ig with
the
same isotype AND dilution (universal negative is stupid). So if your
primary
is an IgG1 from a mouse and you use it at 1:100, your negative control
would
be a non-immune mouse serum IgG1 diluted to 1:100. Running this on
unrelated
material will not show you anything in this case. what it will show you
is
that the detection system you are using is specific to the antibody you
put
on the tissue and not to A) something else in the tissue or B) something
on
the Ig that is not the epitope itself.
Although, If you run the same primary antibody on tissue that you
expect will not react with the primary antibody, this would prove that
the
antibody reaction is specific to the antigenin question. This does raise
the
question of what to do about ubiquitous antigens (like ubiquitin) that
are
actually everywhere. Every cell has

RE: [Histonet] Looking for Aperio or similar Slide Scanner

[Liz Chlipala]

Kathy

We have an Aperio Scanscope XT and scan for a fee

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kathy
Bonness
Sent: Wednesday, May 18, 2011 12:23 PM
To: histonet@lists.utsouthwestern.edu
Cc: Kathy Bonness
Subject: [Histonet] Looking for Aperio or similar Slide Scanner

Hello,

  I am looking for a slide scanner like Aperio to scan my lung tissue
slides at high quality and inclusive of increasingly higher
magnification objectives.  I have the lite version of Aperio to view
slides I scanned in when training at MD Anderson in Houston using their
Aperio and would like to find something similar in the Dallas area if
not at UTSW itself.

  If you have any suggestions of who has this system or one like it that
also allows others to use and/or pay to do so, please let me know.

  Also, if you have suggestions on purchasing one instead or any other
commentaries, I would love to get some feedback.

  Thank you for your time and assistance,

Kathy



Kathy Bonness, PhD.

kathy.bonn...@utsouthwestern.edumailto:kathy.bonn...@utsouthwestern.edu


UTSW  Dallas, TX

Green Center for Systems Biology

Department of Pharmacology

Altschuler/Wu Lab  ND9.214

mailto:kathy.bonn...@utsouthwestern.edu



UT Southwestern Medical Center
The future of medicine, today.
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RE: [Histonet] sections falling off slides

[Liz Chlipala]

Robin

We run quite a few IHC's on goat bone femurs.  We use a very good plus
slide. Dry flat at 40C overnight and let them sit a few days before we
stain, we do not place them in a 60C oven since we find that the
articular cartilage may flip.  We have good success with proteinase K
with staining on the dako stainer for several antibodies.  If we have to
run HIER we use a 70C retrieval for 2 hours and then may choose to
manually stain depending upon the samples rather then placing on the
dako autostainer.  We have done this type of staining on porcine femurs
on 2x3 slides manually.  

I really think tissue adherence is dependent upon good fixation, optimal
decalcification and proper processing, if samples have been
insufficiently decaled or processed you are going to run into problems
with tissue adherence.  I can't comment to the lack of staining
intensity we always pilot a few slides prior to staining the entire
study just to make sure our methods will work for the particular tissues
we are currently working on.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robin
Dean
Sent: Monday, May 16, 2011 12:07 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] sections falling off slides

Hi All,

 

We are trying to do IHC for multiple antigens on goat knees. The
sections
are decalcified FFPE sections and we are using a Dako autostainer. We
are
currently trying air-drying for  1-2 days  and baking of tissue slides
before use in IHC..

The one previous time we tried baking, we noticed a decrease in IHC
signal.
The sections are pretty large, we tried cutting them down, but can't
decrease the size much.  We also  decreased the number of washes and
size of
washes on the Daok, which helped only slightly.  Does anyone have any
suggestions?

 

Thank you in advance.

 

Robin

 

Robin R. Dean, Ph.D.

Senior Scientist  Study Director

Comparative Biosciences, Inc.

786 Lucerne Dr.

Sunnyvale, CA

(408) 738-8060

robin_d...@compbio.com

 

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RE: [Histonet] preference: fat stains

[Liz Chlipala]

Michelle

We use osmium all of the time, we find it works better for image
analysis than the Oil Red O.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Michelle
Perrins
Sent: Friday, May 06, 2011 7:07 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] preference: fat stains

Hello
 
I would like your views on which provides the better result regarding
fat stains.
1. fat stains (Oil red O, etc) on frozen sections
2. osmium tetroxide on formalin fixed tissue
 
I know that osmium is nasty to work with but besides that does it
produce a better result?
 
Thanks
 
Michelle 
 
 
Michelle Perrins
Chief Medical Technologist
Forensic Pathology Services
Division Forensic Medicine 
Faculty Health Sciences
University of Cape Town
 
tel: +27 21 406 6001
fax: +27 21 448 1249
Email: michelle.perr...@uct.ac.za


 

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RE: [Histonet] TNF alpha antibody

[Liz Chlipala]

We have used the antibody from abcam on FFPE mouse tissue with good
success, have not tired it on frozen sections

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Michele
Wich
Sent: Thursday, April 28, 2011 10:08 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] TNF alpha antibody

Can anyone recommend a good TNF-alpha antibody for frozen mouse tissue? 

Any suggestions are greatly appreciated!

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RE: [Histonet] decalcification of articular cartilage

[Liz Chlipala]

We do a lot of IHC staining on bone and cartilage samples and we use 10% formic 
acid for decalcification for immunohistochemistry.  I'm afraid you may not be 
able to turnaround this in a week.  The tissue needs to be adequately fixed 
prior to decalcification, and properly decaled.  I would trim the tissue to 
about 4 mm thick if you can on a saw you might be able to turn this around in  
your time frame, but I have learned from experience if you rush decalcification 
you have a greater chance of running into problems.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Nejat Yilmaz
Sent: Wednesday, April 20, 2011 8:12 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] decalcification of articular cartilage

Dear Members,

We're planning to study sheep knee articular cartilage with histochemistry and 
immunohistchemistry. Proximal parts of the tibia bones fixing in formaldehyde 
at the moment. We need an effective, quick and cartilage tissue protective 
decalcification method. We'll perform only light microscopic examination in 
this study and we have to complete the study within one week. In these 
circumstances what are your advices?
Thanks in advance.

Dr. Necat Yilmaz
University of Mersin
School of Medicine


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RE: [Histonet] processing times for skin specimens

[Liz Chlipala]

Carol

We are a research lab but we have always processed our skin samples on
longer processing cycles, even the punch biopsy samples.  The shaves
might be different, if I wanted to cut down I would start with how you
normally process them and then cut down at 5 minute increments to start
and see how that goes.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Carol
Bryant
Sent: Thursday, April 21, 2011 8:10 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] processing times for skin specimens 

Hello everyone!
What is the shortest cycle you may be using for processing skin
specimens?  Can skin biopsies, shaves, and punches be processed well on
a short run on the VIP 6?  I need to find a successful protocol if
possible to run skins on a 6 hour process, if possible.
Thank you in advance for any input.

Carol


Carol Bryant, CT (ASCP)
Cytology/Histology Manager
Pathology Services
Lexington Clinic
Phone (859) 258-4082
Fax (859) 258-4081
cb...@lexclin.com



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RE: [Histonet] Sad news from Nebraska

[Liz Chlipala]

That has to be an April Fool joke, got ya!!!

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Ian
Young
Sent: Friday, April 01, 2011 12:39 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Sad news from Nebraska

Histonetters,

Those living in Nebraska may want to read this article.
http://www.histobob.com/?q=node/156

HistoBob.com
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RE: [Histonet] HercepTest

[Liz Chlipala]

We use the PT Link unit for Hercept Test

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cindy Bulmer
Sent: Tuesday, March 22, 2011 11:12 AM
To: Histonet
Subject: [Histonet] HercepTest

I understand the HercepTest calls for a waterbath for the epitope retrieval 
process... but,
I'm wanting to know what type of equipment, ie:  waterbath make and model or if 
people are using the Dako PT link module.
 
Cindy

Cynthia Bulmer HT(ASCP)QIHC
IHC Supervisor, CTPL
Waco, TX
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RE: [Histonet] Verhoeff/Masson's Stain

[Liz Chlipala]

John

We do this stain all of the time, but we make up our reagents from
scratch we don't use a kit.  My suggestion is to make up your elastic
stain fresh.  We make up our elastic stain fresh and use it only once
(or for that day) and then discard.  I have attached our procedure in a
different e-mail.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of John
Shelley
Sent: Tuesday, March 22, 2011 11:59 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Verhoeff/Masson's Stain

Hello Histonetters,

I have been asked to work on a project that will require me to do a
Verhoeff/Masson's stain. I have run some samples through the stain from
a protocol that I found on the archives and it is not working to my
satisfaction. I am not getting the nice elastin fibers to show black
like they should. I have looked at the slides just before going into 2%
ferric chloride and it appears to be dull or limited staining. I am
using a kit from PolyScientific and not sure if that might be my problem
or not. My question is, are there another people who are running this
special stain combo and would you be willing to share the procedure and
also who you are buying your reagents from to accomplish the staining. I
appreciate any help with this request. Thanks!!!

Kind Regards!

John J Shelley


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RE: [Histonet] RE: comprehensive list?

[Liz Chlipala]

Vendor sheets may not be accurate we keep an internal log of all of our
antibodies and what species we have tested them in.  So far in our lab
we have found several antibodies that the vendor states works in a
species and it does not in our hands, in these cases we always contact
the vendor, basically for a refund and they have updated their
specification sheets to reflect the change.  We do use protein atlas a
lot for verification of the IHC protocols and tissue staining patterns.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
shive...@umn.edu
Sent: Friday, March 18, 2011 1:06 PM
To: Truscott, Tom
Cc: Histonet; Morken,Timothy
Subject: Re: [Histonet] RE: comprehensive list?

I've also found that occasionally the antigen retrieval method quoted on
a 
vendor's data sheet doesn't unmask antigens as well as another method
tried 
in my initial antibody validation trials with that antibody. So, my
point 
is... any comprehensive list should be used only as a guideline; results
in 
your lab will be affected by the reagents, equipment, and environmental 
conditions you employ in your own lab.

Jan Shivers
UMN Vet Diag Lab


On Mar 18 2011, Truscott, Tom wrote:

 Yes, vendor sheets are good, but not all give enough info, and you may 
have to compare several vendor, and as you say, antibodies only tested
by 
western blot are common in research. Yet, it would be a great money and 
timesaver if there was one site to go to, to see if anyone had tried
these 
antibodies by different methods and could also submit their own results
for 
entry, possibly with links to Journal articles, much like what some
vendors 
do. It could be a quick reference guide to setting up some research or 
double staining methods, or just see if some new request is going to fit

your routine. Someone would have to have more time and computer skills
than 
I. Thanks all for the input, TOM T

-Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Morken, 
Timothy
Sent: Friday, March 18, 2011 10:38 AM
To: Histonet
Subject: [Histonet] RE: comprehensive list?

Tom, 

 Vendor datasheets are the best source of information. The vast majority
of 
clinical vendors antibodies are designed for formalin fixed paraffin 
embedded tissue. Research vendors are all over the map and often only
test 
by western blots or elisa so they may or may not have information about
how 
their antibodies work in fixed tissue.

Tim Morken
Supervisor, Histology, IPOX
UCSF Medical Center
San Francisco, CA, USA


-Original Message-
 From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Truscott, 
Tom
Sent: Friday, March 18, 2011 9:02 AM
To: Histonet
Subject: [Histonet] comprehensive list?

 Hi All, Is there a comprehensive list in one location somewhere that
gives 
information on all or most antibodies on how they work with FFPE or FS
or 
other fixatives, so that we don't have to try to many approaches when we

get a new antibody to try. Thanks, Tom T
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RE: [Histonet] Processing animal fat

[Liz Chlipala]

5mm is pretty thick still.  We process skin and fat and we use a longer
processing cycle.  1 to 1.5 hours per station.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Sherwood, Margaret 
Sent: Thursday, March 17, 2011 1:57 PM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Processing animal fat

My question is directed specifically to veterinary histologists or
histologists
who also do a fair amount of animal processing.  We are having a
terrible time
processing pig fat.  We had problems previously, but thought we had
solved them.
This latest project (pig skin with a lot of fat attached) came out
awful.  The
fat was not adequately processed:  couldn't section it, it just
crumbled.  In
the block, it appears white and crumbly.  The funny thing is some blocks
came
out all right, but most didn't.

PLEASE help!  Let me know how you process your animal fat (sp. Pig)! Is
there a
size issue (we trim it if it is greater than 5mm)?  We have gotten help
from the
histonet before and instituted these suggestions (i.e. let sit in
formalin for
48 hours; put in cassettes in a 60 degree oven for 1-2 hours and then
cool to
room temp. Then process using a 1 hour program).

Any help would be appreciated.

Thanks!
Peggy

Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherw...@partners.org 




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addressed. If you believe this e-mail was sent to you in error and the
e-mail
contains patient information, please contact the Partners Compliance
HelpLine at
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RE: [Histonet] Processing animal fat

[Liz Chlipala]

The laser treatment should not be an issue, we do that type of work all
of the time.  I would trim to about 2-3 mm.  I did notice that you only
have 2 changes of citrisolv a xylene substitute I have not worked with
that one in particular but if I do work with a xylene substitute we use
three changes and we also have 3 to 4 changes of paraffin depending upon
sample size.  For dermis and skin we have a tendency to process longer
but have never experienced any over processing or cooked samples. 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Sherwood, Margaret 
Sent: Thursday, March 17, 2011 2:29 PM
To: Grantham, Andrea L - (algranth); HISTONET
Subject: RE: [Histonet] Processing animal fat

Every station is 1 hour, and our processing program is similar to yours:
we run
the progam overnight, so specimens sit in formalin for a delayed start.
Then
50%, 70% for 30 min. ea.  Then 2x95%, 3x100%, 2xCitriSolv all for 1
hour.  We
end in one change of paraffin. Our specimens are similar in size to
yours,
except we keep the thickness below 5mm.

Any thoughts?  Most of our experimental pig skin/fat are laser treated.
Could
that be a problem? 

Peggy

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Grantham, Andrea
L - (algranth)
Sent: Thursday, March 17, 2011 4:10 PM
To: HISTONET
Subject: Re: [Histonet] Processing animal fat

Peggy,
Is your whole program one hour or is each station one hour?

We had a project here a few years ago that we called the bacon project
because
we had whole chunks of pig skin with an implant and lots of fat in
between the
layers. It was fixed for a couple days in 10% NBF and then processed
with one
hour for each station starting in 70% ETOH and ending in 4 paraffins on
a VIP.
Turned out beautiful. Did trichromes and they were stunning - red, white
and
blue! The sections were big - about 2.5 cm square - took almost all of
the space
in the cassette.

Andi


On Mar 17, 2011, at 12:57 PM, Sherwood, Margaret wrote:

 My question is directed specifically to veterinary histologists or
histologists
 who also do a fair amount of animal processing.  We are having a
terrible time
 processing pig fat.  We had problems previously, but thought we had
solved
them.
 This latest project (pig skin with a lot of fat attached) came out
awful.  The
 fat was not adequately processed:  couldn't section it, it just
crumbled.  In
 the block, it appears white and crumbly.  The funny thing is some
blocks came
 out all right, but most didn't.
 
 PLEASE help!  Let me know how you process your animal fat (sp. Pig)!
Is there
a
 size issue (we trim it if it is greater than 5mm)?  We have gotten
help from
the
 histonet before and instituted these suggestions (i.e. let sit in
formalin for
 48 hours; put in cassettes in a 60 degree oven for 1-2 hours and then
cool to
 room temp. Then process using a 1 hour program).
 
 Any help would be appreciated.
 
 Thanks!
 Peggy
 
 Peggy Sherwood
 Lab Associate, Photopathology
 Wellman Center for Photomedicine (EDR 214)
 Massachusetts General Hospital
 55 Fruit Street
 Boston, MA 02114-2696
 617-724-4839 (voice mail)
 617-726-6983 (lab)
 617-726-1206 (fax)
 msherw...@partners.org 
 
 
 
 
 The information in this e-mail is intended only for the person to whom
it is
 addressed. If you believe this e-mail was sent to you in error and the
e-mail
 contains patient information, please contact the Partners Compliance
HelpLine
at
 http://www.partners.org/complianceline . If the e-mail was sent to you
in
error
 but does not contain patient information, please contact the sender
and
properly
 dispose of the e-mail.
 
 
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RE: [Histonet] Research and Clinical Labs

[Liz Chlipala]

Amos

We are a GLP compliant contract research lab, and we are currently in
the process of applying for a CLIA accreditation.  I don't see why you
would need to have separate equipment, etc.  In our case we will not be
processing standard diagnostic samples but we feel that the CLIA
accreditation will be good for us to have in order to process clinical
trial study samples.  Maybe Haji or someone from Phenopath labs can
comment I believe both of those labs are both CLIA accredited and GLP
compliant.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amos
Brooks
Sent: Monday, March 14, 2011 6:04 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Research and Clinical Labs

Hi,
Let me preface this by stating that I don't think this is a good
idea
and it is not what we are doing here. I have a hypothetical question
though.
What would be the regulatory ramifications of merging a research lab
testing
animal specimens (not regulated by any agencies other than Best
Practices
and OSHA) with a traditional (CAP, JACHO) clinical histology lab. What
equipment, if any, could be shared? In the event of a worst case
scenario
(like an inspector noticing mouse tissue and human tissue on the same
processor at the same time or expired chemicals not labeled research
use
only) which regulations would be violated and how would one find out
potential fines that could be incurred. I have heard that there are labs
that are doing this. What precautions should a lab take if they end up
venturing down this path.

Thanks,
Amos
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RE: [Histonet] VALIDATION of a new clone

[Liz Chlipala]

If it's a different clone then it's essentially a different antibody you
would have to go through the same process as you did initially.

LIz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vickroy,
Jim
Sent: Monday, March 14, 2011 12:17 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] VALIDATION of a new clone

I am wondering what others  are doing when they validate a new clone of
a antibody that you have used in the lab for quite sometime.  When we
introduce a new antibody obviously we go through a pretty extensive
validation process including sampling of around 20 - 30 specimens, some
presumed positive cases and some presumed negative cases.   We also try
to include in the positive cases some that are strongly positive and
others that are weakly positive.

Lately several of our regular antibodies are now no longer available in
the same clone.  I am interested in how different labs would validate
the new clone to be used in the lab.  It would seem to me that the
validation process would not have to be as extensive since we have
already established that the antibody works well in our laboratory.   I
know if it was the same clone but a different lot we would just do a
comparison of the old lot with the new lot, but what about a different
clone?   I realize that the methods are going to be diverse but I would
appreciate hearing from others.


James Vickroy BS, HT(ASCP)

Surgical  and Autopsy Pathology Technical Supervisor
Memorial Medical Center
217-788-4046



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RE: [Histonet] Her2 Gastric cases

[Liz Chlipala]

Margaret

Dako has a bunch of info on Her2 staining in gastric cancer its in the
most recent Connection Volume 15 and they also have a companion document
that covers Her 2 staining and scoring for gastric cancer, just go to
the Dako website and you should be able to view these documents. 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Coppin,
Margaret
Sent: Friday, March 11, 2011 9:54 AM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Her2 Gastric cases

Hello,

 

I am hoping someone can shed some light on whether or not Her2 4B5
(Ventana's antibody) is okay to run on gastric biopsies. We are getting
some client requests for this and I wonder if I should re-direct them
toward the Dako Hercep Test instead.

 

Thank you.

 

Margaret G. Coppin, HT(ASCP)

Technical Supervisor--Immunohistochemistry

 

ARUP Laboratories

500 Chipeta Way

Salt Lake City, UT 84108

(801)583-2787 X3869

copp...@aruplab.com

 

 

 


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RE: [Histonet] Competancies for handling hazardous material

[Liz Chlipala]

One quick correction you may have state reqs, not all states do.  Here
is what I took from Janet and Dick Dapson's book 3rd Edition which is
from 1995.  In 1995 Iowa did not have a state office but the Regional
EPA office number is (913) 551-7050 or (800) 223-0423.  I also thought
it was odd that it was the Colorado Department of Public Health that
inspected me, but when I looked in the book that's who they listed at
the States Hazardous Waste Management Agency.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Friday, March 04, 2011 9:52 AM
To: Jeff Birkner; Podawiltz, Thomas; Fortin, Joyce; Rae Staskiewicz;
Histonet
Subject: RE: [Histonet] Competancies for handling hazardous material

Jeff

Those are the federal Reqs.  You have state reqs that you need to meet
also.  When we were inspected by the Colorado Department of Public
Health they told us that we had to have training and that it did not
matter how many employees that I had since I was now considered a small
quantity generator verses a conditionally exempt small quantity
generator.  EPA is going to be different than OSHA.  With OSHA you have
to have 10 or more employees to be subject to the reqs.  I have less
than 10 employees, but we perform training on everything and have a
considerable safety manual that includes practically everything.  

One other thing, if you are a small business the government will provide
you with assistance you just need to go to the OSHA website, they may
also provide assistance to others.  We had a voluntary inspection from
OSHA.  It took a while to happen since we are not considered a high risk
workplace, but it was helpful.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: Jeff Birkner [mailto:jbirk...@colabserv.com] 
Sent: Friday, March 04, 2011 7:47 AM
To: Podawiltz, Thomas; Fortin, Joyce; Liz Chlipala; Rae Staskiewicz;
Histonet
Subject: RE: [Histonet] Competancies for handling hazardous material

I have reviewed 40 CFR Part 262 and the only training requirements that
I see are the RCRA for Large Quantity generators and those for
university or academic institutions.  I do not see any requirements for
medical laboratories that fall outside of the above
generator/institution types.  Can someone point me in the right
direction if this is not correct?  Thanks!

Jeffrey C. Birkner, CT(ASCP)
Manager, Pathology Laboratory Section
Collaborative Laboratory Services, L.L.C.
1005 Pennsylvania Ave, Suite 102
Ottumwa, IA  52501
641-455-5414
ORHC Extension #3538
jbirk...@colabserv.com
 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Podawiltz, Thomas
Sent: Thursday, March 03, 2011 2:04 PM
To: Fortin, Joyce; Liz Chlipala; Rae Staskiewicz; Histonet
Subject: RE: [Histonet] Competancies for handling hazardous material

We did a self audit about three or four years ago. My area is considered
a small waste storage area and yes there is required training. In New
Hampshire, we can get that training through the state. 




Tom Podawiltz, HT (ASCP)
Histology Section Head/Laboratory Safety Officer
LRGHealthcare
603-524-3211 ext: 3220

From: histonet-boun...@lists.utsouthwestern.edu
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Fortin, Joyce
[joyce.for...@uhsinc.com]
Sent: Wednesday, March 02, 2011 1:21 PM
To: Liz Chlipala; Rae Staskiewicz; Histonet
Subject: RE: [Histonet] Competancies for handling hazardous material

Could I PLEASE have this information, too?  I would really appreciate
it.


Joyce Fortin
Histology Supervisor
Palmdale Regional Medical Center
38600 Medical Center Drive
Palmdale, California  93551
Phone 661-382-5723
Fax   661-382-5747
email:  joyce.for...@uhsinc.com

From: histonet-boun...@lists.utsouthwestern.edu
[histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz Chlipala
[l...@premierlab.com]
Sent: Wednesday, February 23, 2011 6:51 PM
To: Rae Staskiewicz; Histonet
Subject: RE: [Histonet] Competancies for handling hazardous material

Rae

We have, you need standard training, but the individuals who inspected
our lab did not mention compentances.  But our inspection happened
because we needed to switch from a conditionally exempt small quantity
generator to a small quantitiy generator and we then needed to register
with the state, etc.  They primarily

RE: [Histonet] Mouse cardiac myosin antibodies

[Liz Chlipala]

Mark

I took a brief look at the abcam antibody and in particular the review
with the paraffin staining.  If I was purchasing the antibody I would
question the results of that review, since they used a goat anti-mouse
detection system which is not ideal for mouse tissue.  Plus I'm not sure
that the staining pattern is consistent with what I would expect.  I
would expect to see staining of the myofibrils within the cell.  It
appears from the image to be staining nuclei and the outside of the
cell. I would call Abcam to see if they have tested this antibody
internally for paraffin processing or did they completely rely on that
review.  You could also try enzyme retrieval rather than HIER.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mark
Elliott
Sent: Wednesday, March 02, 2011 3:59 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Mouse cardiac myosin antibodies

I have been asked to stain mouse hearts for cardiac myosin. I have tried
the antibody from Abcam (ab50967-Mouse monoclonal to heavy chain cardiac
myosin) with no luck. I am using the Mouse on Mouse kit from Biocare and
it worked well for me for some other mouse antibodies on mouse tissue
but doesn't want to work on this antibody.  I have tried different
retrieval solutions (pH6 and pH9, as well as Biocare rodent Decloaker).
Anyone use this antibody?  Any suggestions for another antibody greatly
appreciated.
 
Thanks
Mark

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RE: [Histonet] Polymer detection kit on rat tissue

[Liz Chlipala]

Anita
 
Since you are working on rat tissue, you have to be careful on what detection 
systems you use.  You will need two different detection systems for your work.  
One for the mouse monoclonal antibodies and one for the rabbit polyclonal.  If 
you want to use a polymer based detection system then here are some basic rules 
you will need to follow.
 
1.  For blocking its probably easiest to use a serum free protein block - many 
vendors have them.
 
2.  For the rabbit polyclonal antibody you can use any vendors anti-rabbit 
polymer, but you can NOT purchase a dual link polymer (one that will detect 
both mouse and rabbit primary antibodies) that will lead to background staining
 
2.  For the mouse antibody on rat tissue if you are dead set on using a polymer 
then you would need to purchase one that is specifically designed to be used 
for mouse antibodies on rat tissue.  Biocare Medical and MaxVision have them, 
there may be others out there these are the two that we have used in our lab.
 
3.  Polymer based reagents are nice but they are expensive.  I don't want you 
to get the empression that I'm knocking polymer based reagents, we use them in 
our lab all of the time.  But there are other methods or detection systmes 
available for use on animal tissues if you are on a budget.  The key to good 
success with animal detection systems if you are going to use a secondary 
antibody rather than a polymer is to make sure that the secondary antibody that 
you purchase has been cross absorbed to the species you are working with. You 
must also check the spec sheets they will normally give you a cross reactivity 
list of tissues that have been tested.
 
Good Luck
 
Liz



From: histonet-boun...@lists.utsouthwestern.edu on behalf of 
amitapan...@torrentpharma.com
Sent: Thu 2/24/2011 3:11 AM
To: histonet@lists.utsouthwestern.edu; histonet-boun...@lists.utsouthwestern.edu
Subject: [Histonet] Polymer detection kit on rat tissue



Dear histonetters,

I am lost in data sheets of  IHC detection kits . Please help me  to
finalize one good detection kit for my purpose.

I am planning to do immunohistochemistry on paraffin embedded rat kidney
tissues for mouse monoclonal and rabbit polyclonal antibody (not  double
labelling).
I am looking on Dako envision, vector immpress, Novolink , the company
like Biocare/ Max bio vision suggest the Rat specific detection kitGot
confused...


Also send me  your opinion on ABC detection versus polymer detection
system.


Thanks in advance for your view.

Amita
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RE: [Histonet] Digital Pathology/Telepathology

[Liz Chlipala]

Andrew

We are a research contract lab and have been scanning slides (WSI) since
2007.  We have an Aperio Scanscope XT.  Our clients have secure remote
access to their images.  We manage our own server, storage (20TB) and
backup locally.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrew
Byrnes
Sent: Wednesday, February 23, 2011 6:35 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Digital Pathology/Telepathology

Dear Histonet,

Please don't take this as spam.  I am just gathering some information...

I am curious to know if any of you are currently working with digital  
whole slide scanners and what your experience has been to date.

Additionally, my company provides telepathology services by putting a  
digital scanner into your lab allowing our pathology group to access  
electronic requisitions and digital slides remotely.  We then return a  
report electronically within 24 hours.  All is stored on our servers  
for secure access from anywhere at anytime.  The idea is to allow easy  
access to our fully sub-specialized pathology group by any  
laboratories that may have a need for more consistent pathology  
coverage or if there are quality issues.  Same concept as  
teleradiology (Virtual Radiologic, Nighthawk Radiology) which is  
implemented in many hospitals throughout the US.  If interested, our  
website is www.accelpath.com

Would love to hear your thoughts on these subjects... digital path and  
telepath.

Have a great day!
Andrew Byrnes
AccelPath





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RE: [Histonet] Competancies for handling hazardous material

[Liz Chlipala]

Rae
 
We have, you need standard training, but the individuals who inspected our lab 
did not mention compentances.  But our inspection happened because we needed to 
switch from a conditionally exempt small quantity generator to a small 
quantitiy generator and we then needed to register with the state, etc.  They 
primarily focused on our waste streams and what chemicals were in the lab.  I'm 
not at work right now, but I'll check tomorrow to see if I have anything that I 
can share with you.
 
Liz



From: histonet-boun...@lists.utsouthwestern.edu on behalf of Rae Staskiewicz
Sent: Wed 2/23/2011 6:19 PM
To: Histonet
Subject: [Histonet] Competancies for handling hazardous material





Has anyone been inspected by the EPA regarding removal of hazardous
chemicals?  It came up in a safety meeting that I should have training and
competencies (beyond general safety training and PPE) for pouring xylene
from staining dishes and processors into accumulation containers. 



Rae Ann Staskiewicz

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RE: SPAM-LOW: [Histonet] TRAP staining kit

[Liz Chlipala]

I'm in agreement with Patsy.  We have found in our hands that the Sigma Kit 
will not work on formic acid decaled bone.  It may work on EDTA decaled bone, 
the reason I say may is that we have gotten variable staining results with EDTA 
decaled bone.  For mouse knees and ankles we have gotten great results with 
EDTA decaled and paraffin processing.  We did modify the protocol a bit.  We 
tried it on EDTA decaled rat ankles and it did not work.  It might be due to 
the length of time in EDTA, I'm not sure, but bottom line it may not work.  At 
least that’s what our experience has been.  We also will use the TRAP antibody, 
we have found that it works in human mouse, and canine so far on formic acid 
decaled and paraffin embedded samples (and again I think length of time in 
decal may impact staining, the canine samples were teeth and surrounding soft 
tissue and it worked ok but not as well).  We have tried this antibody also on 
EDTA decaled mouse bone and it did not work, not sure why but in this instance 
we only received blocks so we did not have any control over fixation, decal or 
processing.  

If you do run into issues there are other markers out there that will stain 
osteoclasts, but are not entirely specific to them.  We have used the following 
antibodies;

Cathepsin K - works on formic acid and paraffin embedded in mouse and rat, but 
it may stain some inflammatory cells also
ED-1 - granted it’s a macrophage marker but it also work nicely to stain 
osteoclasts in rat bone, but again since it is a macrophage marker you are 
going to have to read through that.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Hermina 
Borgerink
Sent: Monday, February 21, 2011 11:45 AM
To: Patsy Ruegg; 'Kim Merriam'; 'Histonet'
Subject: RE: SPAM-LOW: [Histonet] TRAP staining kit

Hi Kim and Patsy,

This protocol works well on EDTA decalcified FFPE tissue samples.


Tartrate-Resistant Acid Phosphatase (TRAP) histochemistry

For Paraffin:
Deparaffinize sections through xylenes and alcohol and bring to running 
deionized water for 5 minutes. 
 
For Plastic:
Deplasticize through three 20-minute changes of 2-methoxyethyl acetate, two 
5-minute changes of acetone followed by running deionized water for 5 minutes.
Make up acetate buffer in the volume needed for the number of slides to be 
stained.

0.2 M ACETATE BUFFER
Distilled water   for 50.0 ml for  
200.0 ml
0.2 M sodium acetate (FW 82.03)  0.82 gm 3.28 gm
50 mM L(+) tartaric acid (FW 230.1) 0.58 gm 2.32 gm
Stir using a magnetic stir bar until dissolved;  pH to 5.0

Following the 5-minute wash under running water, incubate the sections in 0.2 M 
acetate buffer for 20 minutes at room temperature.  After this time has 
elapsed, to this same acetate buffer add:

   for 50.0 ml  
   for 200.0 ml
0.5 mg/ml naphtol AS-MX phosphate 25.0 mg  100.0 mg 
1.1 mg/ml fast red TR salt  55.0 mg   220.0 
mg 

OR

Add the naphtol AS-MX phosphate and TR salt to freshly prepared and preheated  
0.2 M sodium acetate buffer


Incubate the sections from 1 – 4 hours at 37C, monitoring after the first 
hour, until osteoclasts are bright red.  Rinse in distilled water, followed by 
counterstaining in Mayer’s hematoxylin for 1 – 5 minutes (depending on the age 
of the hematoxylin), wash in running water for 5 minutes, air-dry and mount 
with permount.


Ref.  Erlebacher  Derynck J Cell Biol 132:  195 – 210, 1996


September 30, 2003 (Revised)
Hermina Borgerink
Wake Forest University Health Sciences

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patsy Ruegg
Sent: Monday, February 21, 2011 12:46 PM
To: 'Kim Merriam'; 'Histonet'
Subject: RE: SPAM-LOW: [Histonet] TRAP staining kit

Kim if you are asking if TRAP enzyme histochemistry works on formalin fixed
paraffin embedded decalcified bone in my experience it does not.  It works
on smears and cytology slides or GMA embedded bone without decal, it may
work on ETA decaled bone but I do not think it works on ffpe slides.  There
is an antibody to TRAP now you could use on ffpe sections by IHC.  What are
you trying to use it for, osteoclasts???

Regards,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu

[Histonet] IgG and IgM on mouse kidneys

[Liz Chlipala]

Hello all

 

I have a quick question.  Can I perform IgG and IgM staining on mouse
kidneys on FFPE tissue samples?  I thought that this type of staining is
normally completed on frozen sections via Immunofluorescence.  Any
advice would be helpful

 

Thanks

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: [Histonet] Information Needed

[Liz Chlipala]

Pamela

We use a hepa filtered vacuum from Marmed, Inc.  (440) 572-5175

LiZ

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Marcum,
Pamela A
Sent: Friday, February 18, 2011 1:08 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Information Needed 

Hi,

I need information for purchasing a good vacuum with proper biological
filtration for cleaning a cryostat.  We are not buying a new cryostat
and I do know of several with a decontamination feature, it is not in
the budget for the next two years.

Thanks,

Pamela A Marcum
Anatomic Pathology Manager
University of Arkansas for Medical Sciences
4301 W Markham St Slot 502
Little Rock AR 72205
Office: 501-686-5941
Pager: 501-395-1943
Fax: 501-686-7151

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RE: [Histonet] processing histogel

[Liz Chlipala]

Luciana

You process histogel samples essentially the same way you process
regular tissue samples.  I'm assuming you will be dealing with a about a
1 cm cube sample that you want embedded into the histogel and then you
would then slice the histogel cube (with the collagen construct) into 3
mm sections and process to paraffin.  You will need some type of mold to
make the histogel cube.  I would use a peel-away mold that is a bit
bigger than your construct.  If your construct is smaller than the 1cm
cube then you might be able to get away with a cryomold, or embedding
mole.  I would basically use the histogel as the manufacture states,
melt to the correct temp, pour a bit of it into the mold, place your
fixed construct on top of it and fill up the mold with more histogel,
let solidify and then place the entire sample in 10% NBF and then
process to paraffin.  We have dealt with a lot of different constructs
and sometimes you just have work with them a bit to get what you need
out of the paraffin sections.  I would not waste your cultured samples
right away but work out your technique on the construct alone initially.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Luciana
Bastos
Sent: Monday, February 14, 2011 6:06 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] processing histogel



Hello,
We are working with cell culture embedded into three dimensional (3D) of
collagen gel type I.  The samples were fixed in 10% formalin for 24 h.
Even after fixation, the gel used in our preparation was too soft to be
embedded directly in paraffin. To circumvent this problem, we wold like
to dehydrated and embedded the samples in Histogel (Perk Scientific
Inc., Devon, PA, USA).11 V.M. Freitas and R.G. Jaeger, The effect of
laminin and its peptide SIKVAV on a human salivary gland adenoid cystic
carcinoma cell line, Virchows Arch 441 (6) (2002), pp. 569-576. Full
Text via CrossRef | View Record in Scopus | Cited By in Scopus (12)11
However, we need some help to how dehydrated the collagen gel and the
histogel (solutions, concetration, time, ect.) toparaffin-embedded and
stained by haematoxylin-eosin (HE).
Thank you,
Luciana


 
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RE: [Histonet] bar-coded chemical inventory

[Liz Chlipala]

I would be interested in that info too.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Seaton,
Brandon W
Sent: Thursday, February 10, 2011 12:37 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] bar-coded chemical inventory

Anyone out there using barcode systems to inventory chemicals and
organize MSDS, expiration dates, lot numbers, NFPA, etc?  If so, what
system and how well does it work?  In particular I'm curious about the
CISpro system.  Many thanks!

Brandon






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RE: [Histonet] Help with OCT problem

[Liz Chlipala]

Donna

I have seen soft or rather sticky OCT samples in the past.  If you
freeze in isopentane and don't let the isopentane evaporate off the
samples prior to wrapping the sample in foil, that excess isopentane
changes the OCT, it makes it sticky.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Reynolds,Donna M
Sent: Thursday, February 10, 2011 12:39 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Help with OCT problem

Has anyone ever experienced OCT blocks that cut like they are soft. I
have turned the cyrostat down 10 degrees lower than usual and they are
still soft. Really weird part is that I have 20 blocks in this group and
some are soft and others are just fine. Tried changing angle, new blade
nothing helps. When I finally manage to get a decent section and pick it
up on the slide, the OCT around the tissue want even pick up smooth it
wrinkles really bad.
I tried just a block of OCT that I froze on a chuck and it cut great.  I
am clueless as to what is going on.
Samples were frozen by someone else and brought to me to be cut.
Donna Reynolds Core IHC Lab
M.D. Anderson Cancer Center, Houston TX
Research lab
713-192-8106.


Donna Reynolds Core IHC Lab
Dept. Cancer Biology, SRB 1.660
713-792-8106

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RE: [Histonet] IHC validation

[Liz Chlipala]

Joe

If you are following the recommendations from the CAP paper on IHC
standardization then it would be 25 tissues (10 strong positive, 10 weak
to moderate positive and 5 negative). 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joe
Nocito
Sent: Tuesday, February 08, 2011 3:44 PM
To: Histonet
Subject: [Histonet] IHC validation

Greetings Histoland,
I need some help. We are about to switch IHC machines from the
Richard-Allen Axiom to the Ventana Benchmark Ultra. How many slides, per
antibody, do you run for the validation study? We have over 100 primary
antibodies. Normally, when we work up a new antibody, we  start with a
titer. Once that is established, we run 10 cases to check for
specificity. Hopefully we can obtain cases that are really positive,
some weakly positive and some flat out negative. Once that is completed,
we run 10 different tissue types to check for any unexpected
cross-reactivity. 
The ultra holds 30 slides and we are receiving two machines. If we
run 10 slides/antibody, that's going to take a while, not to mention the
number of detection kits that will be used. Do you think 5
slides/antibody is sufficient? I emailed CAP last week for their take
and they never returned my email (I told my medical director to hold
their check for the year and see how fast they respond to that). Ah oh,
don't go down that road Joe, it's unhealthy. What are your thoughts?
Thanks

Joe (JTT)


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RE: [Histonet] IHC validation

[Liz Chlipala]

Nope that's the recommendation for everything, in the paper they state
additional development is required for prognostic markers. Once you have
validated an antibody it only requires 3 tissues when you get a new lot
number:  1 strong positive, 1 weak to moderate positive and 1 negative.
From how I read the article its 25 tissues and then 3 tissues, it does
not talk about slides so it is possible to put multiple tissues on one
slide.  Again these are just recommendations; I do not think that there
is a set standard yet.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: Weems, Joyce [mailto:jwe...@sjha.org] 
Sent: Tuesday, February 08, 2011 4:13 PM
To: Liz Chlipala; Joe Nocito; Histonet
Subject: RE: [Histonet] IHC validation

But that is for receptors, correct? Do you do that for everything? 
Thanks, j 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Tuesday, February 08, 2011 17:58
To: Joe Nocito; Histonet
Subject: RE: [Histonet] IHC validation

Joe

If you are following the recommendations from the CAP paper on IHC
standardization then it would be 25 tissues (10 strong positive, 10 weak
to moderate positive and 5 negative). 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC
PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 fax (303)
682-9060 www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joe
Nocito
Sent: Tuesday, February 08, 2011 3:44 PM
To: Histonet
Subject: [Histonet] IHC validation

Greetings Histoland,
I need some help. We are about to switch IHC machines from the
Richard-Allen Axiom to the Ventana Benchmark Ultra. How many slides, per
antibody, do you run for the validation study? We have over 100 primary
antibodies. Normally, when we work up a new antibody, we  start with a
titer. Once that is established, we run 10 cases to check for
specificity. Hopefully we can obtain cases that are really positive,
some weakly positive and some flat out negative. Once that is completed,
we run 10 different tissue types to check for any unexpected
cross-reactivity. 
The ultra holds 30 slides and we are receiving two machines. If we
run 10 slides/antibody, that's going to take a while, not to mention the
number of detection kits that will be used. Do you think 5
slides/antibody is sufficient? I emailed CAP last week for their take
and they never returned my email (I told my medical director to hold
their check for the year and see how fast they respond to that). Ah oh,
don't go down that road Joe, it's unhealthy. What are your thoughts?
Thanks

Joe (JTT)


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RE: [Histonet] IHC validation

[Liz Chlipala]

Thanks Tim

This is a good way of approaching this. What types of antibodies do you
suggest running the reproducibility tests.  For reproducibility I run
about 3 slides in 3 different runs.  I only run reproducibility tests
for antibody cross reactivity studies (just after protocol development)
and for GLP protocol development.  I don't run all of this for my
routine protocol development, but we keep tract of everything we have an
ongoing log for each antibody and lot number and what conditions they
were stained under along with all of the documentation we keep.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: Morken, Tim [mailto:timothy.mor...@ucsfmedctr.org] 
Sent: Tuesday, February 08, 2011 4:38 PM
To: Liz Chlipala; Weems, Joyce; Joe Nocito; Histonet
Subject: RE: [Histonet] IHC validation

When changing instruments you are validating the instrument, not the
test. For each antibody you just need to run parallel tests in each
instrument showing equivalence. 

However, if you change the instrument and ALSO change the antibody
and/or detection system, Antigen retrieval, etc, then you need to
revalidate the test.

For most antibodies (Class I FDA exempt, ancillary tests) that involves
5-10 positive and negative cases. A small tissue array can suffice. You
should do reproducibility tests for a few antibodies - 5-10 identical
slides on one run, 5-10 identical slides on 5-10 runs.

When changing Class II antibodies (ER, PR, Her2) and/or their detection
systems you will need to run more extensive test. As Liz says, 40
positive, 40 negative. Again, a tissue array is great for that. 

Changing instruments these days is a big deal when a vendor ties you to
their reagents. 

Tim Morken
Supervisor, Histology, IPOX
UCSF Medical Center
San Francisco, CA, USA

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Tuesday, February 08, 2011 3:20 PM
To: Weems, Joyce; Joe Nocito; Histonet
Subject: RE: [Histonet] IHC validation

Nope that's the recommendation for everything, in the paper they state
additional development is required for prognostic markers. Once you have
validated an antibody it only requires 3 tissues when you get a new lot
number:  1 strong positive, 1 weak to moderate positive and 1 negative.
From how I read the article its 25 tissues and then 3 tissues, it does
not talk about slides so it is possible to put multiple tissues on one
slide.  Again these are just recommendations; I do not think that there
is a set standard yet.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: Weems, Joyce [mailto:jwe...@sjha.org] 
Sent: Tuesday, February 08, 2011 4:13 PM
To: Liz Chlipala; Joe Nocito; Histonet
Subject: RE: [Histonet] IHC validation

But that is for receptors, correct? Do you do that for everything? 
Thanks, j 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Tuesday, February 08, 2011 17:58
To: Joe Nocito; Histonet
Subject: RE: [Histonet] IHC validation

Joe

If you are following the recommendations from the CAP paper on IHC
standardization then it would be 25 tissues (10 strong positive, 10 weak
to moderate positive and 5 negative). 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC
PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 fax (303)
682-9060 www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joe
Nocito
Sent: Tuesday, February 08, 2011 3:44 PM
To: Histonet
Subject: [Histonet] IHC validation

Greetings Histoland,
I need some help. We are about to switch IHC machines from the
Richard-Allen Axiom to the Ventana Benchmark Ultra. How many slides, per
antibody, do you run for the validation study? We have over 100 primary
antibodies. Normally, when we work up a new antibody, we  start with a
titer. Once that is established, we run 10 cases to check for
specificity. Hopefully we can obtain cases that are really positive,
some weakly positive and some flat out negative. Once that is completed,
we run 10 different tissue types to check for any unexpected
cross-reactivity. 
The ultra holds 30 slides and we are receiving two machines. If we
run 10 slides/antibody, that's going to take a while, not to mention the
number

RE: [Histonet] Rat lung histology for mast cells and eosinophils

[Liz Chlipala]

Chen

For mast cells you can fix in 10% NBF and be fine with toluidine blue.
We stain with toluidine blue on formalin fixed samples all of the time.
The trick is not to dehydrate the sections, let them air dry and then
mount.  I would stick with formalin fixation that's going to be your
best bet for most specials and IHC stains.  For eosinophils you can
stain with giemsa, or we would run a modified dif-quik stain that worked
well for eosinophils.  For neutrophils I'm not sure but if you are
staining for mouse neutrophils, serotec has a nice antibody.
Macrophages would be IHC for ED-1 also from serotec, CD3 from Dako will
work on rat tissue nicely.  There are other markers that will work on
cell lines.

Good Luck 

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Chen,
Shu-Cheng
Sent: Wednesday, February 02, 2011 11:29 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Rat lung histology for mast cells and eosinophils

Hi,

We are called to support an asthmatic rat lung project and need to stain
for mast cells, eosinophils, neutrophils and possibly other inflammatory
cells, such as Th2 cells etc. Any suggestions you can give us in terms
of fixatives and special stains or IHC are very much appreciated. From
my search, it seems that Carnoy's fixative with toluidine blue is the
way to go for mast cells. But can one clearly differentiate eos from
neuts with this fixative or a formalin fixed HE stained lung section?
If we need to do Trichrome, PAS, May Grunwald Giemsa and HE etc. what
would be the best fixative to accommodate these stains?

BTW, I heard that formalin free Zn-fixative is good for IHC. Will it be
good for all these stains above?

Sorry for so many questions. It shows how little experience I have in
this area.

Thank you,
Shu

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RE: [Histonet] Her2 Fixation Requirement

[Liz Chlipala]

Lori

 

I would think both if you could since the Her2 paper does address those
issues as potentials for problems in testing variation (time to
fixation).  I believe the CAP/ASCO paper states not less then 6 and no
more than 48 hours for fixation (2007 paper).  I believe you also should
document type of fixative if possible; vendor, lot number and expiration
date if you can.  I have pdfs of these documents if you need them.

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
lori.dis...@hcahealthcare.com
Sent: Wednesday, February 02, 2011 12:16 PM
To: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Her2 Fixation Requirement

 

  What is the appropriate time it should be in formalin?  Do you start
your time when it is initially put into formalin, or the start from the
time it was dissected?

 

Lori A Disher

Fawcett Memorial Hospital

Port Charlotte,  FL   33952

lori.dis...@hcahealthcare.com

 

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[Histonet] EGF IHC

[Liz Chlipala]

Hello everyone

 

Does anyone out there have a good EGF (not EGFR) antibody that works
well in formalin fixed paraffin embedded samples - species is human

 

Thanks

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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[Histonet] slide labeler advice

[Liz Chlipala]

Hello again

 

We are in the market for a slide labeler.  We currently have the
SlideMate from Thermo Scientific.  We have had it for about 2 years now
and we are just not happy with the quality and consistency of the
printing.  We are a contract research lab and not a clinical lab.
Entering data for each slide is not an option.  The programming that
comes with the Slide Mate is nice, except that no one has really spent
the time to train us on it, we have figured it out a bit. We would want
a labeler that we could create formats for different sample types, so
all we would have to do is put the animal ID in and the printer would
print all of the slides needed.  Would like barcoding capabilities just
incase we move to that in the future.  Any advice or suggestions are
appreciated.  Vendors welcome also.

 

Thanks in advance. 

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: [Histonet] stains for visualizing new bone growth

[Liz Chlipala]

Robin

I'm not sure how accurate this is, but we do a lot of massons trichrome
staining in rat muscle pouch studies (they inject into the muscle pouch
something that will induce bone formation) and we have noticed that new
bone formation has a tendency to stain blue rather than red.  You could
also try a Goldners trichrome too.  That is supposed to stain new bone
formation.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robin
Dean
Sent: Tuesday, February 01, 2011 12:45 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] stains for visualizing new bone growth

Does anyone know of a good stain to use to clearly show new bone growth
other than von Kossa stain?

Would appreciate any suggestions anyone might have.

 

Thank you,

 

Robin

Robin R. Dean, Ph.D.

Senior Scientist  Study Director

Comparative Biosciences, Inc.

786 Lucerne Dr.

Sunnyvale, CA

(408) 738-8060

robin_d...@compbio.com

 

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RE: [Histonet] ventilation and exposure

[Liz Chlipala]

Curt

We fill our bottles in the hood.  They are emptied into a vented
flammable cabinet that contains a 55 gallon drum.  You know you can do
your own chemical badges for both xylene and formalin.  I like to run a
couple TWA (8 hour) exposures and then I badge a lot of STEL (15
minutes) exposures on what I think are worse case scenarios or where I
think individuals have exposure for a short period of time, like
changing the tissue processor, or loading the tissue processor, changing
the stainer, manually coverslipping, grossing in tissues, etc.  

If you are a small business OSHA has support services.  They will come
to your lab and perform an audit and review your safety practices (non
punitive) and give you a report where they think you can improve.  It
was helpful to us.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Curt
Tague
Sent: Friday, January 28, 2011 10:23 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] ventilation and exposure

Yesterday I asked for some direction on ventilation firms to evaluate my
lab
and the fumes. I think I've got some decent leads but I have another
quick
question, does any change the processor bottles, formalin, alcohols,
xylem/subs, inside a ventilated hood?

 

Thank for your input,

 

Curt 

 

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RE: [Histonet] Cytoplasmic staining with Ki67 in frozen sections

[Liz Chlipala]

James

I believe the nuclear staining pattern that you describe is normal for
Ki-67 in the spleen, not sure about the cytoplasmic or particulate
staining.  

Since you viewing with fluorescence the particulate staining may be
autofluorescence of red blood cells. Normally our primary antibody
incubations are only 30 minutes, 2 hours at RT seems a bit much. Did you
try multiple dilutions of the primary?

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of James S.
Sent: Thursday, January 27, 2011 2:36 AM
To: 'histonet@lists.utsouthwestern.edu'
Subject: [Histonet] Cytoplasmic staining with Ki67 in frozen sections

HI All,

I've just been trying out a Ki67 antibody from Abcam (rabbit polyclonal)
on some frozen mouse tissues. Staining looked ok in the colon with
nuclear localisation in the cells of the basement membrane. However,
what I'm really interested in is the spleen and the staining here was a
mess! There was some nuclear staining especially in germinal centres and
scattered around the white pulp but the red pulp contained clusters of
cells with cytoplasmic staining only (I don't know if this is real
staining, non-specific staining or an artefact), also there was bright
particulate 'staining' all over the red pulp but not the white.

Frozen sections were cut (10um), air dried overnight, fixed in acetone
(10mins), blocked with 5% normal goat serum, Ki67 Ab for 2hrs room temp,
secondary was goat anti rabbit AlexaFluor 488 (Invitrogen - clean on its
own).

I thought this Ki67 staining was going to be easy!!

Any suggestions?

Thanks
Sonya


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RE: [Histonet] Oil Red O for FS on Muscle

[Liz Chlipala]

Allison

We make up our Oil Red O from scratch same day we use it, let it stand 10 
minutes, and then filter with a Millipore Stericup 0.22µm, GP Express Plus 
Membrane, 250ml Receiver Bottle, catalog number: SCGPU02RE under vacuum.  That 
seems to help a bit with the background and we are working with a really clear 
solution. We have not run muscles but aortic root and liver on mouse and rat.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Akemi Allison
Sent: Wednesday, January 26, 2011 10:16 AM
To: histonet
Subject: [Histonet] Oil Red O for FS on Muscle

Happy hump day!

Does anyone have a good procedure for Oil Red O for FS on Muscle.   
The  procedure the lab I am working with is having a great deal of  
problems with their muscle biopsy panel.  I am trouble-shooting some  
of the issues for the 1-Step Trichrome 3.4 pH, NADH, AT Pase,  I am  
working with them with pH issues.

The Oil Red O is very problematic.  They are mixing before use and  
filtering with 42 Watman paper, but there is a lot of residual  
background on the slides.  Also, they were not fixing the sections  
with 37% formaldehyde, even though the procedure calls for it.  Could  
you share who you get the stain from also.  Your assistance is  
greatly appreciated.

Akemi


Akemi Allison BS, HT (ASCP) HTL
Director
Phoenix Lab Consulting
Tele: 408.335.9994
E-Mail: akemiat3...@yahoo.com

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RE: [Histonet] Consult Fees for IHC

[Liz Chlipala]

Joe

Keep in mind that that what you charge you need to take into
consideration the taxes you will be responsible for paying.  Most
consultants are paid a simple fee and the company paying you will not
take out any taxes. So you need to account for that.  I have known quite
a few individuals who got themselves in a bind when it came to tax time.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joe
Nocito
Sent: Wednesday, January 19, 2011 3:59 PM
To: Histonet
Subject: [Histonet] Consult Fees for IHC

Greetings histo land,
Hope everyone is having a great New Year. I have been asked to consult
on IHC to a local firm. They asked me what my fees are. I told them we
can talk about that later. I have no idea what to charge these people. I
want to be fair, but I don't want to give this knowledge away (an y'all
thought that this was just a petty face). Any ideas for the Texas area.
This would be telephone and on site consulting for a research firm.
Thanks in advance.

Joe
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RE: [Histonet] coverglass

[Liz Chlipala]

That's who we use and we don't have any problems, with have the Tissue
Tek Glas coverslipper

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer
Campbell
Sent: Wednesday, January 12, 2011 11:35 AM
To: Histonet
Subject: [Histonet] coverglass

I am looking for vendors for coverglass for an automated coverglasser.

I want one where the coverglasses don't stick together. We are currently
using Mercedes as our vendor.

Any feedback out there for ThermoFisher?

-- 
Jen Campbell
Supervisor of Technical Services
Muhlbauer Dermatopathology Laboratory
P: 585.586.5166
F: 585.586.3137
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[Histonet] Feulgen Stain for DNA

[Liz Chlipala]

Hello Everyone

 

I have tried to find this answer on the web and in references but I need
some help.  With respects to the Feulgen stain, how sensitive is it? Can
it detect smaller fragments of DNA? Does it have to be double stranded
or will single stranded stain as well?  Thanks in advance.

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: [Histonet] Research charges - long reply

[Liz Chlipala]

Mary

Mary

We are a private contract lab and do not charge for recuts or repeats
that are related to quality issues.  We run a lot of bone samples
through our lab and if there is a fold in the lesion area we recut the
block at no charge to the client.  All of our slides are QC'd and we
will repeat anything we think we need to prior to shipping the study
back to the client. 

If the client is not happy with the work, (which happens rarely) we will
repeat at no cost. We are a quality lab that stands by that so we need
to follow all of these steps.  I feel it helps us maintain our clients,
our clients know that we have their best interest in mind and that we
are honest with them. We work with them all of the time to develop
grossing, processing, sectioning and staining protocols that are
specific to their tissues.  Some of our basic charges to process and
stain one block with HE we charge $16.00 to $20.00, for hand processing
we charge $25.00 per block and that includes an HE, for unstained
slides on plus slides $4.50 specials range from $16.50 to $29.50
depending upon the type of special.

A lot of the tissues we work with need to be sectioned to a specific
area, for example on mouse knees we need to section to the center of the
knee joint, or with mouse or rat eyes we need to section in the area of
the optic nerve head.  All of it is very detail orientated.  You can
sometimes tell if you are in the correct area by just looking at the
tissue in the block, then we would section, stain and review, if we were
not in the correct area we would section again, stain and review.  This
took us a lot of time and we never would charge for those additional
sections that we took.  What we do now is that we have nice student
microscopes (leica we spent around $1200.00 for each of them) next to
each microtome so as the techs cut into the block they pick up and can
review the unstained sections, this has helped us out a lot. We have
less recuts now.  The samples that we need to fiddle with a bit on
trimming are the ones that we charge $20.00 per block, like bone and
eyes.

For those individuals who want to stand by you while you section through
their block and only collect the lesions I would either charge them an
hourly rate or state that they have to pay for each unstained slide and
stained slide - you should be doing this already.

Researchers sometimes have limited budgets, but they should be paying
for what you provide to them.  You have a budget that you have to work
with, and they need to be aware of that, your time and supplies cost
money too.  You can work deals out, like having them pay for supplies
for their projects. You sometimes need to get creative.

I hope this helps

Liz 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mary
Lloyd
Sent: Thursday, January 06, 2011 4:35 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Research charges

Would anyone like to share their charges for research.  I am interested
in these charges.  
Routine processing
Hand processing
HE 
Unstained slides
Routine Special Stains (PAS,GMS etc.)
I would also like to know policies on charging for repeat work.  If a
researcher is interested in an area(bone) that is difficult and needs to
be recut over and over again because of lifting and folding in the area
of interest.  After using many different techniques I have resolved the
problem but some researchers demand more and more for free.
  Also I have a couple of researchers that stand with me while I am
cutting through the block to keep the lesional slides only in their
blocks.  It takes around 45 minutes per block.  Is their any
compensation for the time I spend with the researcher. 

I put through some new policies and prices but my pathologist says I
need documentation of those charges.
 Thanks Mary

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RE: [Histonet] Lab chairs

[Liz Chlipala]

Sharon

We got chairs from VWR - catalog number 80086-440 VWR(r) Contour(tm)
Self-Skinned Urethane Chairs, we got these back in 2007 or 2008 and they
have held up great, they are comfortable and clean very easily.  They
might be a bit pricey.  I think I paid around $150.00 to $200.00 for
each back when I bought them and that was with special pricing from my
rep.  I see on line now that my pricing is $277.00 per chair and close
to $400.00 if you do not have any discounted pricing with VWR.  But
overall they are great chairs.  As histotechs we sit almost all day long
depending upon what task we are performing so at the time I wanted to
purchase good supportive chairs that I know we would be able to clean
and keep for a while.  So far it has been a good investment.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Sharon.Davis-Devine
Sent: Wednesday, January 05, 2011 9:19 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Lab chairs

We are getting new lab chairs for our Histology deparment and are
wondering what other labs out there have used.  We want something that
has good ergonomic support, is not cloth, because of the paraffin and is
not too expensive. Any suggestions or ideas are greatly appreciated.
Thanks.

Sharon Davis-Devine, CT (ASCP)
Cytology-Histology  Supervisor
Carle Foundation Hospital
Laboratory and Pathology Services
611 West Park Street
Urbana, Illinois 61801
217-383-3572
sharon.davis-dev...@carle.com

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[Histonet] FW: ASCP Action Alert - Physician Signature Requirement _FYI

[Liz Chlipala]

FYI - saw alot about this today, just got this e-mail from ASCP

Liz

-Original Message-
From: American Society for Clinical Pathology [mailto:a...@site1.ascpmail.org]
Sent: Wed 12/22/2010 2:42 PM
To: Liz Chlipala
Subject: ASCP Action Alert - Physician Signature Requirement
 
 http://portal.mxlogic.com/images/transparent.gif 

 


Physician Signature Requirement Burdens Labs, Jeopardizes Patients 

ASCP urges you to act before the Jan. 3 deadline 

Recently, the Centers for Medicare and Medicaid Services (CMS) finalized its 
Medicare Physician Fee Schedule final rule for the 2011 calendar year. The rule 
includes a troubling policy change requiring a physician's or qualified 
nonphysician practitioner's (NPP) signature on requisitions for clinical 
diagnostic laboratory tests paid under the clinical laboratory fee schedule 
(effective Jan. 1, 2011). CMS defines a requisition as the actual paperwork, 
such as a form, which is provided to a clinical diagnostic laboratory that 
identifies the test or tests to be performed for a patient. Currently, a 
physician signature is required only on orders for laboratory services. 

ASCP believes the new rule could adversely affect patient care and complicate 
the provision of the laboratory services. In cases where a signature from the 
ordering physician or NPP is absent, laboratories could be left scrambling 
trying to obtain the signature. 

Late Breaking News Rule: Recently CMS has partially agreed to a delayed 
implementation request from ASCP and other members of the clinical laboratory 
coalition organizations. CMS will delay implementation of the rule for three 
months. During this period, ASCP will continue to pressure CMS to withdraw its 
new proposal. 

ASCP urges you to contact CMS and tell them of your opposition to the policy 
change. 

Contact CMS before the Jan. 3 Comment Deadline to maximize the effectiveness of 
your voice. 

Read more and take action today . . . 
http://emessage.chicago.ascp.org/emessageirs/servlet/IRSL?v=4l=1r=10644m=22015e=2
 

  


 

Tell a friend:
Not everyone receives ASCP's Action Alerts, so please forward this message to 
your peers and co-workers!


 http://www.chicago.ascp.org/pics/email/logoASCP-final-2736.jpg 

2010 American Society for Clinical Pathology
33 West Monroe Street, Suite 1600, Chicago, IL 60603
312.541.4999 
~ i...@ascp.org 

 http://portal.mxlogic.com/images/transparent.gif 

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RE: [Histonet] Reg: Beta-2 minroglobin IHC

[Liz Chlipala]

Robin

Have you tried other retrieval methods, its best to normally try to stay
away from HIER on bone but sometimes you have to use it.  If you have
not already tried other retrieval methods I would try no retrieval,
proteinase K and then trypsin or pronase, my favorite is proteinase K.
Since the samples have been decaled we have sometimes noticed in our
hands that the nuclei can  get chewed up a bit with enzyme retrieval
so if you notice that you may need to back off on the time of retrieval,
just as long as you do not loose staining intensity.  If this antibody
will not work with any other retrieval method then you will have to use
HIER retrieval.  Instead of retrieving at 95 or 100C or even higher I
would go with 70C for several hours (2-3) you are going to have to pilot
this. That's what we do here for any sample has a tendency to lift or
distort when retrieved (bone, skin).  You may then be able to run the
slides on a stainer but again if you see tissue detachment you are going
to have to run them by hand.

We use a good superfrost plus slide such as histobond for bone and if we
are running into problems with tissue lifting we let the unstained
slides sit for a few days even a week before we stain and we find that
that helps also.

There is a human mitochondria antibody that we have used in the past in
rat samples, but I can't remember what retrieval method works.  There is
also a human nuclear antibody which works well on frozen sections but
can be tricky on paraffin, we have not had much success with it on
paraffin sections.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Robin
Dean
Sent: Wednesday, December 08, 2010 9:20 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Reg: Beta-2 minroglobin IHC 

Hi all,

 

We are trying to use beta-2 microglobulin IHC staining to ID human cells
in
goat joints that were engrafted with human cells some time ago and
having
nothing but trouble. Tissue was decalcified in immunodecal,
formalin-fixed
paraffin-embedded tissue. Beta-2 MG antibody is a polyclonal rabbit X
human
beta-2 antibody from Acris, and we are using DAKO Envision-HRP -con.
secondary with DAB as chromogen.

IgG isotype controls and DAB alone controls look clean.

. Goat joints are falling off the charged slides with
heat-induced
citrate buffer antigen retrieval so went to enzymatic retrieval to keep
them
on the slide.

o   Does enzymatic antigen retrieval damage this epitope?? (previous
successful uses of this antibody were with citrate antigen retrieval)

o   Any suggestions on how to keep sections on slides? Sections are kind
of
large, but pathologist wants that size.

. Didn't get hardly any staining at 2.5 ug/ml of the beta-2MG
antibody, but at 5 ug/ml everything stains including all of goat joints.


. Are there other or better stains that are used for identifying
human cells engrafted into other species (label uniformly most human
cells)?
I saw mention of some mitochondrial antgen antibodies but no specifics.

. Does Beta-2  antibody label only human cells? Seems like there
are
homologous markers in other species that may label. 

Currently we aren't using any blocks except peroxidase block. Thought we
might try a CAS block to see if that will prevent sticking? To non-human
tissue.

 

Any suggestions or help with these problems will be greatly appreciated.
We
have to get the stain to work and are running out of ideas.

 

Robin 

 

Robin R. Dean, Ph.D.

Senior Scientist  Study Director

Comparative Biosciences, Inc.

786 Lucerne Dr.

Sunnyvale, CA

(408) 738-8060

robin_d...@compbio.com

 

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RE: [Histonet] Decalcification

[Liz Chlipala]

Try surface decaling the samples in the block.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Sherwood, Margaret 
Sent: Wednesday, December 08, 2010 11:41 AM
To: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Decalcification

We have small arteries that were FFPE and sectioned that should have
been
decacified before processing.  (The investigator did not indicate as
such when
submitting them).  Cutting was extremely difficult and the sections, as
you can
imagine, had terrible knife marks and chatter.  Of course, they are
important
(for a paper)!  

Is there any way tissue, that has been embedded, can be decalcified at
this
point?  Or some other way to treat the block so that we can get better
sections?

Thanks for all your help.

Peggy

Peggy Sherwood
Lab Associate, Photopathology
Wellman Center for Photomedicine (EDR 214)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114-2696
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-1206 (fax)
msherw...@partners.org 




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RE: [Histonet] neurodegeneration stains?

[Liz Chlipala]

Anjum

Those are the two that I'm familiar with.  I'm not aware of any others.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of ANJUM
PARKAR
Sent: Tuesday, November 16, 2010 8:55 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] neurodegeneration stains?

Hi all,
I am trying to characterize neuronal degeneration and was wondering if
there
are any fluorescent markers out there to stain for dead/dying neurons? I
have
used Silver stain and am currently trying to optimize the Fluro Jade C
marker
too. But I need some more and the literature seems limited on this.
Thanks

--
Anjum 


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RE: [Histonet] Cheap slide scanner for rat brains?

[Liz Chlipala]

Caroline

The older models of the Nikon scanner for kodachromes had an attachment that 
would scan glass slides, I'm not sure about the new ones but we had one back in 
the late 90's early 2000's that would hold a slide and we could scan that.  You 
could also take some low mag images on a microscope with a camera and them 
merge the images in adobe elements.  It will work but the method can be a bit 
crude and you have to watch your illumination through the entire process.  I 
have even used a regular digital camera and taken images of the glass slide, 
that worked too.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Caroline Bass
Sent: Friday, October 22, 2010 11:50 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Cheap slide scanner for rat brains?

Hello Everyone,

Does anyone have a good way to get low magnification images from slides? I
have some DAB stained rat coronal sections that I would like to digitize.
Basically, I want to have a picture of the entire section. It doesn¹t have
to be high resolution, just enough to make out the basic structures. Can
someone recommend a way to do this? I¹ve used a flat bed scanner in the
past, and I know some folks use an actual film slide scanner. What I¹m
interested in are tips for finding a solution, particularly what to avoid,
and model numbers that people have used. I don¹t want to keep buying
scanners until I found one that works.

And obviously I¹d like to keep the expenses to a minimum.

Finally, has anyone tried this scanner? It¹s a little pricey for me, but I
do like the idea behind it, although I wonder if it¹s just one of their
stock scanners that they have repainted, added an adapter and jacked up the
price for. It looks identical to other scanners that they sell for $300.

http://www.meyerinst.com/html/oem/pse4/

 
Thanks,

Caroline
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RE: [Histonet] Recommendations for decal solution

[Liz Chlipala]

I like 5 to 10% formic acid

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lewis,
Patrick
Sent: Friday, October 22, 2010 2:06 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] Recommendations for decal solution

Hi guys,

 

Can anyone recommend a good decal solution.  I have some bone marrow and
trachea tissues for paraffin sectioning and I want to decal them. 

Thanks

Patrick

 

 

Patrick Lewis

Research Associate II-Bench| Infections and Prematurity

Seattle Children's Research Institute

206-884-1115  OFFICE 

000-000-  PAGER

000-000-  CELL 

206-884-7311  FAX

patrick.le...@seattlechildrens.org

OFFICE  1900 9th Avenue Seattle, WA 98101

MAIL  M/S C9S-8, Seattle, WA 98101

WWW seattlechildrens.org http://seattlechildrens.org/ 

 



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RE: [Histonet] Amputation transport

[Liz Chlipala]

Try mopec

 

-Original Message-
From: Fleming, Jackie M jackie.flem...@allina.com
Sent: Wednesday, October 13, 2010 7:59 AM
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Subject: [Histonet] Amputation transport

Does anyone have a transport/ carrier for transporting amputations.? We
have specimens funneled into one site for grossing, and need to
transport amputations AK and BK. Thanks for any help!


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[Histonet] ECM stain for growing cartilage

[Liz Chlipala]

Hello Everyone

 

Is anyone out there aware of an ECM stain that can be used on growing
tissue culture cells in a hydrogel.  What I'm really looking for is a
live stain I can use on the whole gel and see with a light microscope.
I want to get a general idea of the amount of ECM that is growing.  So
it could just be a general bulk ECM (proteoglycan or collagen) stain
that will show a contrast from the gel.  And I want to be able to return
the gel to the incubator and do the stain again multiple days later.  I
have searched extensively for something that would do this and I have
come up with nothing.  Any advice is appreciated and thanks in advance

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: [Histonet] Bone IHC

[Liz Chlipala]

We lower the temp of retrieval to 70C for 2 hours and have good success
with that.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vanessa
J. Phelan
Sent: Thursday, October 07, 2010 1:14 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Bone IHC

Hi everyone,

I was wondering if anyone has any tricks on how to get bone sections to
stop lifting off the slide through the IHC process? I leave them in the
oven for quite a while to make sure they are baked on, however after
antigen retrieval  (pressure cooker for 20mins) most of the boney part
of the tissue comes off and the marrow and muscle stays put! The
sections are cut onto superfrost plus slides.

Any help would be much appreciated, thanks.

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RE: [Histonet] TUNEL

[Liz Chlipala]

We use the Roche Kit with good success.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Vanessa
J. Phelan
Sent: Friday, September 03, 2010 2:27 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] TUNEL 


Hi everyone,

I am hoping you can help me I am looking for some advice on how to do
TUNEL staining (fluorescent or enzymatic), kits recommended/ protocol.
Any help would be greatly appreciated.

Thanking you in advance

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RE: [Histonet] Collagen Type II antibody

[Liz Chlipala]

Joel

We use an antibody we purchase from MD Biosciences - its quite expensive
but we find it works really well for us on multiple species with
chondroitinase digestion.

Protocol:  Rabbit anti Human Collagen Type II
Clone: Polyclonal 
Vendor: MD Biosciences
Catalog Number: MD20211
Species: Rabbit

Liz


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Joel
Reichensperger
Sent: Monday, August 02, 2010 11:30 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Collagen Type II antibody

  I am in need of a good collagen Type II antibody that could be used in

both rat and human samples. We would prefer a polyclonal antibody but 
might settle for a monoclonal. Anyone have any good suggestions? We will

be staining the samples with DAB. Thanks in advance.

Joel Reichensperger

-- 
Joel Reichensperger
Researcher II
Southern Illinois University
Plastic Surgery Institute
jreichensper...@siumed.edu
217-545-7309 (Office)
217-545-1824 (Fax)


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RE: [Histonet] FW: anti-Ly-6G (aka GR-1 ) IHC question

[Liz Chlipala]

Brett

We have not used that antibody but we have used the following antibody
from Serotec with good success.

Protocol:  Mouse Neutrophil Immunohistochemical Stain
Clone: 7/4
Vendor: Serotec
Catalog Number: MCA771
Species: Mouse
Isotype: Rat IgG2a

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Connolly, Brett M
Sent: Friday, July 30, 2010 11:34 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] FW: anti-Ly-6G (aka GR-1 ) IHC question 


Resending - 


 Does anyone have experience with any anti-mouse LY-6G antibodies on
 FFPE mouse tissue for staining neutrophils??
 
 Looking for antibodies and protocol suggestions.
 
 Thanks,
 Brett
 
 Brett M. Connolly, Ph.D.
 Molecular Imaging Team Leader
 Merck  Co., Inc.
 PO Box 4, WP-44K
 West Point, PA 19486
 brett_conno...@merck.com
 T- 215-652-2501
 F- 215-993-6803
 
 
 
 
Notice:  This e-mail message, together with any attachments, contains
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RE: [Histonet] Procollagen

[Liz Chlipala]

Mary

We use the one from Abcam ab64409, it works really nice

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mary
Abosso
Sent: Wednesday, June 30, 2010 10:21 AM
To: Histonet
Subject: [Histonet] Procollagen

All -
 
We are currently using Pro-COL1A1 (N17) and was wondering what others
are using for a Procollagen for derm applications?
 
Mary
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RE: [Histonet] Canine bone sectioning trouble

[Liz Chlipala]

Overall your fixation, decalcification and processing cycle is too
short.  The sample size is really big, why do you need it so thick?  I
would first of all let the samples fix longer 24 to 48 hours or I would
trim them so that they are about 3-5 mm in thickness. Either way I like
fixing bone samples for 24 to 48 hours.  I'm not that accustomed to
using RDO decal it is hydrochloric acid based and is quicker but even
bone that size would take a while to decal.  We normally use a formic
acid based decal and a sample size that large my take up to 2 weeks to
decal.  Also to process a sample that size I would be at 4 to 6 hours a
station.  We frequently process porcine and goat knees in 2 x 3 blocks
the samples are quite thick about a cm in thickness and we will process
them at 4 to 6 hours a station.

My recommendation would be to decal the larger cube of bone for a period
of time until you can section through it.  I would then cut a 3-4 mm
thick piece off the cube and process it at 1 hour per station.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Schneider,Lynda S
Sent: Tuesday, June 29, 2010 10:18 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Canine bone sectioning trouble

Hello out there...

We are having trouble sectioning canine bone.  The samples are bone
marrow cubes approximately  2cm thick and 1in wide.  They were fixed for
15 hours in 10% NBF and decaled for 30 - 45 mins in RDO rapid
decalcifier.  They were faced in and then surface decaled again for
about 30 mins.  When sectioned, much of the marrow was missing and the
bone was torn and shredded.  We thought that maybe the samples had not
been fixed sufficiently so refixed overnight in 10% NBF again.  The
samples were then reprocessed as they had been originally.  The
processing schedule used was: 70% EtOH 30mins, 80% EtOH 30mins, 95% EtOH
x 2, 30mins each, 100% EtOH x 2, 30 mins each, xylene x 2, 45 mins each,
paraffin x 4, 40 mins each. Again they were faced in and decaled for
another 30 mins or so.  This time as soon as the sections are placed on
the water bath (38 degrees) they explode and/or come apart so severely
sections can almost not be picked up.  If sections are even obtainable
they are of horrible quality.  Does anyone have any suggestions? Thank
you so much in advance!

Lynda
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RE: [Histonet] cryojane question

[Liz Chlipala]

Emily

You can put more than one section on a slide if you need to, but in our
experience it does not work as nicely as depicted all of the time, it
can be a bit tricky to work with on undecalcifed bone and harder
tissues.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Emily
Sours
Sent: Friday, June 18, 2010 8:10 AM
To: Kim Merriam; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] cryojane question

Histonetters,

I just looked up what a cryojane was, and it's pretty neat!
Does anyone else use this?
The one flaw seems to be that you can only put one section on a slide
(or at
least that the way it's depicted here:
http://www.instrumedics.com/cryojanetapetransferprocess.htm )
which makes it pretty time consuming.  Also does the uv step interfere
with
in situ protocols? I guess not since the DNA/RNA is already transcribed
and
fixed and therefore wouldn't be mutated.


Emily


Towns are like people. Old ones often have character, the new ones are
interchangeable.
--Wallace Stegner, Angle of Repose
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RE: [Histonet] ISH on histogel cell pellets

[Liz Chlipala]

Kim the histogel should not interfere with the IHC staining, I'm not sure about 
the ISH

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kim Merriam
Sent: Friday, June 18, 2010 7:09 AM
To: Histonet
Subject: [Histonet] ISH on histogel cell pellets

Hi Everyone,

We have some cells that we have processed and embedded in histogel (thanks to 
everyone for the protocols that were sent to me a couple of weeks ago).

We are planning to do IHC and ISH on these pellets.  Will the histogel 
interfere with the ISH procedure at all?

Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 


  
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RE: [Histonet] how to prevent foldings on femoral head cartilage tissue

[Liz Chlipala]

Sometimes placing them on a hot plate at about 60C will help get out the
folds, the paraffin needs to melt and the sections need to turn clear
then take it off the hot plate.  If you leave it too long on the
hotplate the articular cartilage may fold over on itself.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Reuel
Cornelia
Sent: Thursday, June 17, 2010 11:10 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] how to prevent foldings on femoral head cartilage
tissue

Hello histonetters especailly hard tissue group
I have a  pig femoral head bone tissue embedded in paraffin and I have a
hard time getting rid of the folding problem. I tried to remedy by
lowering my temperature to 38 C and putting them in 5% alcohol before
placing them in water bath I still have a lots of folding formation on
some areas of the cartilage. Is there any other technique to remedy this
problem. I appreciate your help. Thank you.

reuel



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RE: [Histonet] Antibody Validation - long response

[Liz Chlipala]

Bottom line it's not the vendors responsibility to validate their equipment or 
antibodies in your lab. Some vendors may help you do this, but ultimately the 
lab needs to validate the equipment and IHC in their lab.  The vendors normally 
calibrate the equipment prior to shipment and once they set the instrument up 
in your lab, they should be able to provide you with the documentation that 
states that they calibrated the instrument.  Your instruments need to be 
calibrated prior to being validated. 

As far as your scanner goes some vendors can provide validation, but it's at a 
cost and that cost is not cheap depending upon what you actually want 
validated.  If you are using the scanner and associated algorithms for analysis 
then you need to validate that separately.  There are several steps required to 
validate a scanner - 1.  you validate the scanner 2.  if you are using a 
database to store your images then that also may need to be validated and 3.  
if you are using algorithms that provide you with data then those algorithms 
need to be validated.   

For example prior to running a validation protocol on a tissue processor its 
needs to be calibrated for temperature.  All of your major equipment needs to 
be on a calibration schedule.  We calibrate all of our instruments once a year 
and validation is completed only once unless we change the instrument location 
or how we use the instrument. Pipettors are calibrated every 6 months.  All 
instruments are validated it may just be a one pager for the basic lab 
equipment but instruments like the tissue processor, slide staining, IHC 
stainer and scanner require written protocols some of these are 80 pages in 
length and go into great detail.  

The same goes for your antibodies.  Antibodies are validated initially with 25 
tissue samples (10 strongly positive tissues, 10 moderate to weakly positive 
tissues and 5 tissues that have no reactivity) This type of validation is 
required for routine antibodies, prognostic markers such as Her-2, ER and PR 
require additional tissue samples.  New lots require 3 tissue samples one 
strongly positive on moderate to weakly positive and one negative.  If you 
change the antibody source or detection system or retrieval it needs to be 
validated again - This information comes from the paper Standarization of 
Immunohistochemistry from CAP its available on line - I have a copy if you need 
it.  There are also new guidelines for ER/PR and a new article on validation of 
ER/PR in the June issue of Archives of pathology from CAP.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, June 15, 2010 8:50 AM
To: histo...@pathology.swmed.edu; teri.hall...@midmichigan.org
Subject: Re: [Histonet] Antibody Validation

Teri:
You are right about the validations you propose although I am not surprised 
that your vendor does not think it is necessary. They are in the business of 
selling and you are in the business of assuring the high quality of your work 
to obtaining the most accurate work for patients' sake.
There is where the difference resides. Ignore your vendor and keep validating 
your protocols.
René J.

--- On Tue, 6/15/10, teri.hall...@midmichigan.org 
teri.hall...@midmichigan.org wrote:


From: teri.hall...@midmichigan.org teri.hall...@midmichigan.org
Subject: [Histonet] Antibody Validation
To: histo...@pathology.swmed.edu
Date: Tuesday, June 15, 2010, 7:55 AM


I am being questioned by our vendor as to why we need to validate our
automated immunostainer and image analysis instrument. They would like
documentation pertaining to the requirement of validation and the number
of specimens utilized for validation.  I am requesting that each
antibody be validated on the instrument against a previously validated
instrument. Additionally, I am requesting that each new lot of antibody
be validated upon receipt against previously ran specimens. This would
also apply to the image analysis antibodies. (Her2 has been validated by
FISH.) The vendor has apparently polled users in the area and this is
not a standard protocol, therefore the request for documentation. 

I think it is pretty clearly stated by CAP in the Quality Management In
Anatomic Pathology. Any other suggestions?

Teresa Hallada BS, MT/CT (ASCP)
Pathology Lead
MidMichigan Health - Gratiot
teri.hall...@midmichigan.org
989.463.1101 ext 3423

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RE: [Histonet] Mounting Medium for Immunofluorescence

[Liz Chlipala]

I think that the propidium iodide will stain the nuclei or DNA, similar
to DAPI, so this mounting media will counterstain the nuclei and its
visible in the FITC wavelength (488), while if you conterstain with DAPI
it is excited with a different wavelength, therefore it might interfere
with the FITC portion of your stain.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie
Colbert
Sent: Thursday, May 20, 2010 12:13 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Mounting Medium for Immunofluorescence

We normally coverslip our immunofluorescence slides with a permanent
aqueous mounting medium.  By mistake, today they were coverslipped with
a mounting medium that we use for coverslipping our FISH slides.  This
mounting medium is described as Mounting medium for fluorescence, with
propidium iodide.

Will this make a difference when reading the slides??

 

Laurie Colbert

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[Histonet] RE: PBA for Immunofluorescence

[Liz Chlipala]

We just use Dako's serum free protein block for IF.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: Nails, Felton [mailto:flna...@texaschildrens.org] 
Sent: Thursday, May 20, 2010 1:29 PM
To: Liz Chlipala; Laurie Colbert; histonet@lists.utsouthwestern.edu
Subject: PBA for Immunofluorescence

Concerning Immunofluorescence, where are you getting your (PBA) Protein
Blocking Agent from? I use to order it from Thermo Shandon but they no
longer carry it. 

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Thursday, May 20, 2010 1:49 PM
To: Laurie Colbert; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] Mounting Medium for Immunofluorescence

I think that the propidium iodide will stain the nuclei or DNA, similar
to DAPI, so this mounting media will counterstain the nuclei and its
visible in the FITC wavelength (488), while if you conterstain with DAPI
it is excited with a different wavelength, therefore it might interfere
with the FITC portion of your stain.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC
PO Box 18592 Boulder, Colorado 80308 office (303) 682-3949 fax (303)
682-9060 www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie
Colbert
Sent: Thursday, May 20, 2010 12:13 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Mounting Medium for Immunofluorescence

We normally coverslip our immunofluorescence slides with a permanent
aqueous mounting medium.  By mistake, today they were coverslipped with
a mounting medium that we use for coverslipping our FISH slides.  This
mounting medium is described as Mounting medium for fluorescence, with
propidium iodide.

Will this make a difference when reading the slides??

 

Laurie Colbert

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RE: [Histonet] weekend fixation

[Liz Chlipala]

I was talking to Peggy Wenk over the weekend at the MSH meeting and they
had a paper that was published regarding fixation and ER/PR staining
sensitivity etc.  The biggest problem that they reported is
underfixation is much worse than over fixation.  I think a minimum of 10
hours of fixation demonstrated good results and that intensity of
staining started to decrease but not by much at 48 hours.  I would be
more concerned over underfixation than overfixation.  Also the new ER/PR
guidelines state its acceptable to have samples in fixative for 72
hours.  Maybe Peggy can post the link to this paper.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of LINDA
MARGRAF
Sent: Thursday, May 20, 2010 1:04 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] weekend fixation


 Histonetters:
Here's a message I was asked to post..

Dear Colleagues,
 I  have the following question concerning tissue processing. We do a
lot of IHC work on NF fixed tissue. To standardize and minimize the
effect of NF fixation, we fixate the tissue always for 24h. This is of
course a problem for tissues taken on Friday. In the past, we asked our
technicians to come on Saturday to embed the tissues in paraffin.
Unfortunately, this is not possible anymore, and that is why I need your
advice. What would you suggest ? 1) to leave the tissue in NF until
Sunday evening and start processing, or 2) to keep the fixation time (24
hours) and leave the tissue in alcohol 70% until Sunday evening and then
start processing.
 Thanks for your advice.
Kind regards,
Wim.
 

Prof. dr. Wim Van den Broeck, DVM, MSc, PhD
Cell biology and Histology
Department of Morphology - Faculty of Veterinary Medicine
Ghent University
Salisburylaan 133, B-9820 Merelbeke, BELGIUM



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RE: [Histonet] IHC Validation on new instrument

[Liz Chlipala]

We perform our entire validation process as a new piece of equipment.
Our validation protocols are quite extensive, up to about 85 pages long
on each piece of major equipment, at least that's what it was for our
new prisma stainer and glass coverslipper.  We perform an
installation/operational qualification protocol or an IOQ.  If we move
the instrument we also do the same thing, we already have the protocol
written which takes most of the time we just execute it again.  We are a
GLP lab so we work off a Validation Master Plan that basically tells us
how we are going to validate each piece of equipment in the lab.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie
Colbert
Sent: Wednesday, May 19, 2010 9:00 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Validation on new instrument

What do others do when validating a new model of a piece of equipment -
same manufacturer, same basic staining process, but an updated version
of the equipment?

 

I've been told the protocols should be the same and that we only need to
run three controls with three different but similar protocols to
determine what looks best.  Do you all think that is thorough enough, or
would you run actual patient cases and compare old and new equipment?  I
don't see where the CAP checklist refers to new equipment - just new
antibodies and new antibody lots.

 

Laurie Colbert

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RE: [Histonet] PPE

[Liz Chlipala]

I require gloves when sectioning only because it helps eliminate
epithelial floaters, with respects to embedding the tech can choose to
or not to use gloves.  No safety goggles are required.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cindy
DeRiso
Sent: Wednesday, May 19, 2010 12:21 PM
To: Histonet@lists.utsouthwestern.edu
Subject: [Histonet] PPE

Does any one use or require gloves when cutting and embedding? How about

safety goggles when embedding?
Thanks in advance-
Cindy DeRiso
Yale University Pathology

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RE: [Histonet] IHC Validation (again)

[Liz Chlipala]

Laurie

I'm not aware of a particular question, but I would believe you would
have to perform some validation steps for each antibody.  I would
approach it the same way you approach validating new lots of antisera.
The CAP paper on standardization of IHC recommends 25 different samples
when you initially validate an antibody - 10 samples that have high
levels of target antigen, 10 intermediate to low levels and 5 negative.
To revalidate new lots they recommend only 3 tissue samples - 1 high, 1
med to low, and 1 negative.  Granted this only applies to routine
markers.  For prognostic markers such as ER/PR and Her2 then additional
samples need to be tested - there are new guidelines for ER/ER out and
the new recommendations for validation for ER/ER are briefly reviewed in
the CAP Today April issue.  Guidelines for validation of ER/PR will be
published in the June issue of Archives of Pathology and Laboratory
Medicine.

Liz 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie
Colbert
Sent: Wednesday, May 19, 2010 2:01 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Validation (again)

Can anyone tell me if there is a specific question on the CAP checklist
that addresses revalidation of antibodies when starting up a new IHC
stainer?

 

Laurie Colbert

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[Histonet] Article for Er/PR vaidation

[Liz Chlipala]

Hello everyone

 

I have been trying to locate this article on line and I'm either
challenged or its not there.  

 

Its from the Arch Pathol Lab Med (in press)  it says in press so I don't
know what that that means but its not available off the Journals web
site

 

Fitzgibbons, P. L. , D. A. Murphy , M. E. H. Hammond , et al.
Recommendations for validating estrogen and progesterone receptor
immunohistochemistry assays. Arch Pathol Lab Med (in press). 

 

Does anyone have a copy of this that they are willing to forward to me,
the new ER/PR guidelines that just came out reference this article for
validation, even through at the end of the article there is a statement
---  there is no universal acceptable procedure for validating tests
any help would be appreciated.

 

Thanks in advance

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: [Histonet] Thanks for bone cutting suggestions - and anti-Rat CD3

[Liz Chlipala]

Dako has a rabbit polyclonal that works in rat.  The other thing with
the bone, is place the slide with a section on a flat hotplate (60 C)
until it turns clear - not too long or the articular cartilage will
flip.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Connolly, Brett M
Sent: Tuesday, May 11, 2010 11:38 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Thanks for bone cutting suggestions - and anti-Rat
CD3

Thanks to all for your suggestions for cutting cross sections of
cortical bone. I have passed them along.

Now, what is the latest on CD3 IHC in FFPE rat tissues. I saw Ray
Koelling's suggestion in the archives using an Ab from BD Biosciences.
Is it cat # 550295?

What others if any do you suggest? HEIR ? protocol tips?

Thanks,
Brett

Brett M. Connolly, Ph.D.
Molecular Imaging Team Leader
Merck  Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
tel. 215-652-2501 fax. 215-993-6803
brett_conno...@merck.com

 
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RE: [Histonet] CBG BIOTECH RECYCLER

[Liz Chlipala]

Nirmala

We have the CBG recycler also.  We initially started recycling both
alcohol and xylene, but we now only recycle alcohol.  We ended up with
so much recycled xylene that we could not use.  Also you can not use the
recycled xylene on the last xylene station in the tissue processor, or
for cleaning.  It just did not work out for us for the xylene.  We
rotate our xylenes and alcohols weekly so we are always putting on one
fresh absolute and one fresh xylene, there was never a place for us to
use the recycled xylene, so we ended up with lots of it.  If you change
your entire tissue processor then you could put a recycled xylene in
place of your first xylene and then use fresh xylene for the last
station.  We do recycle the alcohol.  We get about 95% alcohol out of
the recycler - we test it for percentage of alcohol via a hydrometer and
for contamination with xylene by adding water to a small portion of it.
We only use it for 95% or less so we make up our 50%, 70% and 80%
alcohol from the recycled alcohol, these solutions do go on our tissue
processor and in the staining set up and they seem to work just fine.
To be honest we do not keep track of how many times the alcohol has been
recycled we just keep recycling it.

As for disposal we have a really cool flammable cabinet that houses a 55
gallon drum, the drum is on rollers so it can be moved easily. All of
our waste goes in there its picked up whenever it is full.  We use
Source/AET Environmental to dispose of all our liquid waste.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
sris...@mail.holyname.org
Sent: Tuesday, May 04, 2010 9:05 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] CBG BIOTECH RECYCLER

Hi All,

Needs some information on waste of xylene and alcohol.

We are currently using a CBG recycler in our lab.  We have been told
that 
the clean alcohols and xylenes should not go in the recycler since it 
contains paraffin. 
Also the observation is, the alcohols put in the machine is percentage 
wise less than what is put in.  In other words the recycled alcohols are

used as 95% or less.  Our cytology department does not used the recycled

alcohols or xylenes since they claim to have staining issues.
Does any one out there has developed a standard of howmany times the 
alcohols and xylene can be recycled?  Do you have a company pick up your

waste after a couple of recycles. 
Who helps out with the waste disposal and are they poured into 55 gallon

drums and hauled away?

Any assistance will be appreciated.

Thanks

Nirmala Srishan
Holy Name Medical Center
Teaneck NJ 07666







Holy Name Medical Center is the recipient of:

Magnet Recognition for Excellence in Patient Care, American Nurses 
Credentialing Center

100 Best Places to Work in Healthcare, Ranked Fourth Nationally by
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Awards for Emergency, Outpatient and Inpatient Service Excellence, J.D. 
Power

Distinguished Hospital Awards for Clinical Excellence, HealthGrades

Excellence Awards for Stroke, Gastrointestinal and Pulmonary Care, 
HealthGrades

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Chest Pain Center Accreditation, Society of Chest Pain Centers

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Department 
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RE: [Histonet] Responses to IHC CAP Validation question

[Liz Chlipala]

CAP does have guidelines, it's a paper that was published a few years
back, its right on the CAP website.  I have it if anyone needs it.  I'm
not sure they use it for their inspections but it covers how to validate
an antibody.  How many samples to run, what paperwork needs to be kept,
etc.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
thisis...@aol.com
Sent: Wednesday, April 28, 2010 12:47 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Responses to IHC CAP Validation question



The following is one respone I rec'd:
 
1.  I asked CAP who told me that they do not currently have a guideline
on
validating but that they
recommend what is in the following book:
Quality Management In Anatomic Pathology, Promoting Patient Safety
Through Systems Improvement and Error
by Raouf E. Nakhleh, MD  Patrick Fitzgibbons, MD editors
sold by CAP !
Chapter 8- Quality Management in IHC
That is what we follow.
I. Get a new antibody and optimize it with your positive control.
II. Once optimized you need to run it on cases expected to be positive
(how many?)
a suffient size ...
III. Must also be run on cases expected to be negative. (how many?
IV. In a situation where you cannot expect a lot of cases or such a
case has
never been presented in your lab, then you must say just that.
(ex. some of the hormones we just use a pituitary)
 
 





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[Histonet] tape coverships

[Liz Chlipala]

Hello all

 

I have a question on tape coverslips.  We received a couple tape
coverslipped slides in for scanning and the tape is completely removed
from the slide.  I would normally just coverslip with glass but the
tissue is on the tape coverslip and not on the slide.  How can I re
attach the tape to the slide, can I use xylene and regular mounting
media?

 

Thanks in advance

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: [Histonet] tape coverships

[Liz Chlipala]

Thanks it worked

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: Mike Pence [mailto:mpe...@grhs.net] 
Sent: Wednesday, April 21, 2010 3:11 PM
To: Liz Chlipala; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] tape coverships

Just put the slide in xylene and coverslip with the tape containing the
tissue. The you could put a couple small drops of mounting media on for
good measure.
Mike

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Liz
Chlipala
Sent: Wednesday, April 21, 2010 3:45 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] tape coverships


Hello all

 

I have a question on tape coverslips.  We received a couple tape
coverslipped slides in for scanning and the tape is completely removed
from the slide.  I would normally just coverslip with glass but the
tissue is on the tape coverslip and not on the slide.  How can I re
attach the tape to the slide, can I use xylene and regular mounting
media?

 

Thanks in advance

 

Liz

 

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC

Manager

Premier Laboratory, LLC

PO Box 18592

Boulder, Colorado 80308

office (303) 682-3949 

fax (303) 682-9060

www.premierlab.com

 

 

Ship to Address:

1567 Skyway Drive, Unit E

Longmont, Colorado 80504

 

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RE: SPAM-LOW: Re: [Histonet] Cd31 in pigs

[Liz Chlipala]

Patsy

How do you order that rat anti-mouse CD31.

Thanks

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Patsy Ruegg
Sent: Saturday, April 10, 2010 10:40 AM
To: 'Jan Shivers'; 'Jean-Martin Lapointe'; histonet@lists.utsouthwestern.edu
Subject: RE: SPAM-LOW: Re: [Histonet] Cd31 in pigs

Yea you can use F8 and SMA for vessel lining cells but those will not pick
up all the developing endothelial cells like cd31 or cd34.

There is a new rat anti mouse CD31 from Japan, I wonder it if works on pig,
I will try it and see, it works great on mouse tissue ffpe.

Regards,

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jan Shivers
Sent: Friday, April 09, 2010 10:24 AM
To: Jean-Martin Lapointe; histonet@lists.utsouthwestern.edu
Subject: SPAM-LOW: Re: [Histonet] Cd31 in pigs

Von Willebrand Factor (Factor VIII) is also what I use to stain pig 
endothelium.

Jan Shivers
Senior Scientist
Pathology Teaching Program
Histology/IHC/EM Section Head
University of Minnesota
Veterinary Diagnostic Laboratory
1333 Gortner Ave.
St. Paul, MN  55108
612-624-7297
shive...@umn.edu

(Confidentiality Notice: This message, together with any attachments, is 
intended only for the use of the individual or entity to which it is 
addressed and may contain confidential or privileged information. If you 
think you have received this message in error, please advise the sender and 
then delete this message and any attachments immediately.)


- Original Message - 
From: Jean-Martin Lapointe jm.lapoi...@accellab.com
To: histonet@lists.utsouthwestern.edu
Sent: Friday, April 09, 2010 10:59 AM
Subject: [Histonet] Cd31 in pigs


Hi Joel,
we have tried a few antibodies for CD31 in FFPE tissues in pigs and have had

no luck. We currently use an antibody to vWF to stain pig endothelium. If 
someone has a working technique for CD31 in swine, I'd be happy to hear 
about it.

Jean-Martin
__
Jean-Martin Lapointe, DMV, MS, dACVP
AccelLAB Inc
Québec, Canada  J7H 1N8
jm.lapoi...@accellab.com



Date: Fri, 9 Apr 2010 09:40:00 -0400
From: Joel Israel jo...@mcclainlab.com


Does anyone have a procedure that WORKS for cd31 in pigs?  I can't seem
to get it to work no matter what I do.  Any help will be greatly
appreciated.  Thanks.- joel



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RE: [Histonet] 72644.18148...@web111105.mail.gq1.yahoo.com

[Liz Chlipala]

Thomas

Just a question, what would you consider an unnecessary CAP regulation?


Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Thomas
Jasper
Sent: Thursday, April 01, 2010 12:46 PM
To: Andrew Burgeson
Cc: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] 72644.18148...@web05.mail.gq1.yahoo.com

I'm inclined to agree with you Andrew.  Seems to me that CAP has become,
unintentionally (I'd like to believe) something of an unsavory, bullying
sort of entity.  I'm not certain about all the factors involved, but I
think a few things have definitely contributed to all of this CAP
negativity.

First of all - CAP was (is?) considered the accrediting gold standard.
That's pretty heady stuff...possible ego inflation potential...high
horse attitude...elitism?  Not saying that was the plan, just an
unfortunate and unintended consequence.  I'm sensing a power trip here,
it's partly human nature (I guess) and it can definitely suck!

Secondly - as I recall, CAP got egg on their face a few years back in
the DC/Baltimore area I believe.  Someone can correct me if I'm wrong or
provide more specific details.  Anyway, the gist being - someplace
passed their CAP inspection and someone (an employee I believe)
contacted another regulatory agency because there was NO way this place
should've passed CAP.  When this all came to light CAP had to respond,
and as can often be the case, the response was overly compensatory.

I'm sure a lot of folks out there know what I mean.  Now CAP issues
confusing and sometimes unnecessary regulation changes and additions.
And of course we're all aware of the super secret surprise
inspections.  I'm not even sure of half the other hidden agendas and
possible ulterior motives.  Control issues, tarnished pride, bruised
egos and all conveniently cloaked in a drive for the best possible
patient care...who could argue with that standard?

During my previous employment, we sweated the details and worked
diligently to achieve our 1st CAP accreditation circa 2000.  I have to
admit, I did/do like the regulation format.  Having a question asked,
determining if it applies to your service, and then answering yes or no.
By taking it from there and doing things on the up and up, most any lab,
that's honest and conscientious should have the realistic expectation of
passing CAP.  That was then, it seems to longer be that way.  As I
mentioned, confusing language and reg. additions/changes, along with CAP
inspectors and their agendas have all been to the detriment of the
accreditation.

I was trained to look at CAP as peer review.  In my experience, many
times this was not the case.  Many CAP inspecting teams wants to make
the inspectees (if that's a word) something of a clone, carbon copy or
version of the inspecting team's service.  This is another huge problem
and causes a lot of strife, hard feelings and red tape at and after the
summations.  The regulations, ideally, should be interpreted in the most
objective way possible.  Again, maybe it's human nature, but it seems
that people can't help being overly subjective re: interpretations of
any number of CAP regs.

I used to work with a pathologist that regularly attended the CAP
committee meetings.  At times I would bring issues to him I thought
relevant to CAP.  I don't recall the specifics but I do know they were
of a practical nature from a technical viewpoint.  I basically got the
brush-off and was led to believe that CAP wasn't interested in the
technical viewpoint and he wasn't going to bother with it.  This may
be a stretch in logic on my part...however, I can't help but think if
CAP would listen to technical folks as well as MDs, they'd be in a
better position right now.

I'm not inclined to throw the baby out with the bath water.  I think CAP
accrediting was established with good intentions. Somehow things have
gotten out of hand, and some have gone horribly wrong.  I think things
like QIP are good, although I've heard complaints about that as well.
I'm feeling lucky these days because CAP isn't in my life.  But my
attitudes and mind-set have most certainly been shaped by my CAP
experiences.  Please remember this is my opinion only, I am not perfect
and am only interested in practical application of sensible regulations
for optimal patient care.

Regards,
Tom Jasper
Histology Supv.
CORPS


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrew
Burgeson
Sent: Thursday, April 01, 2010 10:27 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] 72644.18148...@web05.mail.gq1.yahoo.com

Sheesh is right, J.

CAP 

RE: [Histonet] Incubation of Ab more then 1 hour in autostainer

[Liz Chlipala]

We have done up to an hour but not longer, if I were you I would have
two antibody steps and just to add some more reagent after an hour or
1.5 hours

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
Margaryan, Naira
Sent: Thursday, March 18, 2010 1:39 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Incubation of Ab more then 1 hour in autostainer
Importance: High

Hi Histonetters!

I have a stupid question but I Have to ask.

Does anyone perform an Incubation of Ab that required more then 1 hour
(2-3 hours) in autostainer? Does autostainer keep slides wet or slides
sometimes are getting dry? 

I know that slides should not be dry in any step of IHC.

Thank you very much!

Naira

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RE: [Histonet] CD3 on mouse

[Liz Chlipala]

Dako's rabbit anti-CD3 cross reacts with mouse

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Mauger,
Joanne
Sent: Wednesday, March 17, 2010 1:31 PM
To: Emily Sours; histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] CD3 on mouse

Hi All,
Does anyone know a CD3 antibody that works well on FFPE mouse tissue?
Thanks in advance,
Jo

Joanne Mauger HT(ASCP)QIHC
Children's Hospital of Philadelphia

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RE: [Histonet] tissue processing

[Liz Chlipala]

Sheri
 
Your processing cycle is too long.  We process 20 minutes per station for mouse 
tissue only 2 absolutes and 2 xylenes and 3 paraffins. Even 3 absolutes and 3 
xylenes at 20 minutes a station we find over processes the tissue.
 
Liz



From: histonet-boun...@lists.utsouthwestern.edu on behalf of Sheri Kelemen
Sent: Wed 3/17/2010 4:05 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] tissue processing



Hi everyone,
I need some advice.
I have been doing tissue processing (on an old Shandon Hypercenter),
embedding and sectioning for 15 yrs. and never really had any problems. 
I recently processed mouse tissues on a brand new tissue processor
(Leica ASP300S). When I went to cut 5 micron sections the tissues
(spleen, heart, aorta and lung) were very dry and brittle.  They made
what looked like sawdust after each cut.  Soaking them in cold ammonia
water helped some; but they still did not cut nicely.

Here is my protocol:

I perfusion fixed the mouse with fresh 10% Neutral Buffered Formalin
(NBF).  Then I removed the tissues and cut them in smaller pieces (no
more that 5mm thick) and put them in 10% NBF for 24 hrs., then washed
with PBS and stored in 70% ethanol (made with PBS) for 2 days before
processing (only because I didn't have time to process till then).
My processing protocol is as follows:
70% ETOH x1 1hr.
95% ETOH x2 1hr each
100% ETOH   x3 1hr each
Xylene x3 1hr each
wax x3  45 min each  60°C

Can any one tell me why they think my tissues are so so dry.  What could
I change for the next time I do this?

Thank you.

Sheri Kelemen
Research Associate
Cardiovascular Research Center, Temple University
3500 N. Broad St.
Philadelphia, PA 19140
215-707-3170 work
skele...@temple.edu

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RE: [Histonet] Saffron/Eosin as a counterstain for HE???

[Liz Chlipala]

Its hematoxylin, ploxine and saffron.  Believe it or not it was the
stain that we used at my first job back in 1973.  We also fixed in
Zenkers too.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of sheila
adey
Sent: Tuesday, March 16, 2010 1:04 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Saffron/Eosin as a counterstain for HE???


Hello,

Our pathologist said that he say HE slides with a counterstain that had
some amount of saffron in it?

Is anyone familiar with this? 

Sheila Adey HT MLT 


  
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RE: [Histonet] PPE's embedding and cutting

[Liz Chlipala]

We wear gloves to section, not necessarily for safety reasons but to
eliminate squamous cells on the slides.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
rick.garnh...@memorialhealthsystem.com
Sent: Monday, February 22, 2010 4:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] PPE's embedding and cutting


Anyone in histology land required to wear all PPE's to embed and cut?


Rick Garnhart HT(ASCP)
Memorial Health System
Histology Supervisor
1400 E. Boulder St.
Colorado Springs, CO 80909
Cell: 719-365-8357
Ph:  719-365-6926
Fax: 719-365-6373
rick.garnh...@memorialhealthsystem.com



Mission: To provide the highest quality health care
Vision: To create an outstanding health system where patients heal and
people thrive
Values: Compassion - Integrity - Quality - Respect - Teamwork

www.memorialhealthsystem.com

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RE: [Histonet] static

[Liz Chlipala]

Why don't you ask them to sit down at scope and show you what they are
seeing?  That's the best way to approach any staining or sectioning
issues.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tana
Badalamenti
Sent: Thursday, February 11, 2010 11:22 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] static

Pathologist say that they SEE static on h...@e slides. Does anyone know
what that is and how to remedy it? Thanks, Tana Southern Pathology,
Covington, La.


  
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RE: [Histonet] Help...

[Liz Chlipala]

Fawn

CAP has specific quidelines, its right there in the questions, did you
do what that question is asking and have you documented what was done.
They tell you right in the check sheet what needs to be completed.  Test
validation must be performed on a minimum of 25 cases (they recommend
25-  100) and they tell you how to validate too.  There is also an
article that you can get on the CAP website on IHC standardization. I
have a pdf of it here if you need it.

LIz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Fawn
Bomar
Sent: Thursday, February 11, 2010 12:07 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Help...

Does anyone have a protocol or procedure for validating the Her2
antibody?  We are trying to get ready for a CAP inspection and I'm not
sure if what we did is ok or not.

Thank you in advance
Fawn
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Thank you

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RE: [Histonet] gurney cover

[Liz Chlipala]

I'm not sure of a plastic transparent one, but MOPEC sells the covers.
This goes way back in the early 90's but I think they have a person that
makes them for them, so they may possibly be able to make you one that
is transparent.  Its worth a try and a phone call.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Jennifer
MacDonald
Sent: Monday, February 08, 2010 12:42 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] gurney cover

Does anyone know of a transparent plastic cover that goes over a
gurney?. 
We use cadavers in the anatomy lab and would like to display the cadaver

but prevent students from touching and evaporation during practical
exams.
Thank you,
Jennifer
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