Re: [Histonet] Sections sticking to tweezers

[Caroline Miller via Histonet]

Yes +1 to the chilling of the forceps, especially when you are just
starting out. I often kept two pairs - one I was using and the other
chilling. Also agree about turning your waterbath down a little.

good luck!
Caroline

On Mon, May 8, 2023 at 6:35 AM Rosa, Taylor via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi Ken,
>
> Try chilling the tips of the forceps (I usually keep mine on the ice bath)
> - as long as they are cold, they shouldn't stick to the paraffin.
>
> Thanks,
>
> Taylor C. Rosa, MS
> Research Scientist I
> Pathology Services | Charles River
> 4025 Stirrup Creek Drive, Suite 150, Durham, NC 27703
> P: 919.206.7041 | F: 919.206.7001
> taylor.r...@crl.com | www.criver.com
>
> -Original Message-
> From: Paula Sicurello 
> Sent: 06-May-2023 10:57 PM
> To: Ken M ; Ken M via Histonet <
> histonet@lists.utsouthwestern.edu>
> Subject: Re: [Histonet] Sections sticking to tweezers
>
> Hi Ken,
> A few questions:
> 1. What is the melting point of the paraffin you use?  2. Is there a
> specific reason the temperature of your water bath is 38.5 degrees?  3. Do
> your forceps have any type of imperfections? Bumpy, or ridged? Something
> that could snag on the sections?
> If there isn't a specific reason for the water bath temperature to be that
> low (cutting brain sections), I'm thinking that is playing a large part in
> your sticky sections.
> It is common to have the water bath 5 - 10 degrees below the melting point
> of the wax. It helps the sections stretch out from the compression that
> happens when being cut.
> Let's see what others have to say about your sticky situation.
> Paula Sicurello
> On Sat, May 6, 2023 at 8:27 AM, Ken M via Histonet<
> histonet@lists.utsouthwestern.edu> wrote:Can anyone tell me why my
> sections are sticking to my tweezers and turning into one long string of
> snot when I try to separate them? I have a good pair of curved stainless
> tweezers and I am trying both the front and the back and tapping sharply
> while allowing the tweezers to open at the separation point. I can separate
> one or two, then it sticks and pulls up 7 or 8 sections into one long slimy
> string. My water bath is set at 38.5 and I am sectioning at 4-5 m. Even
> when I go to 6 it still does it. Any ideas?
> Ken___Histonet mailing
> listHistonet@lists.utsouthwestern.eduhttp://
> lists.utsouthwestern.edu/mailman/listinfo/histonet
>
> Paula Sicurello
>
>   On Sat, May 6, 2023 at 8:27 AM, Ken M via Histonet<
> histonet@lists.utsouthwestern.edu> wrote:   Can anyone tell me why my
> sections are sticking to my tweezers and turning into one long string of
> snot when I try to separate them? I have a good pair of curved stainless
> tweezers and I am trying both the front and the back and tapping sharply
> while allowing the tweezers to open at the separation point. I can separate
> one or two, then it sticks and pulls up 7 or 8 sections into one long slimy
> string. My water bath is set at 38.5 and I am sectioning at 4-5 m. Even
> when I go to 6 it still does it. Any ideas?
>
> Ken
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Re: [Histonet] research questions

[Caroline Miller via Histonet]

I would say it depends on the project. I would always strive to get
controls from the same species, but sometimes that is just not possible. If
the marker's cellular or tissue location is easily recognizable then Ok
with the different species, but if it was a diffuse and hard to interpret
marker then I would try harder to get an in-species control.

I don't think I would use a different species control to determine ideal
concentration / protocol for the main cohort. I would get it going on the
control and run the cohort and tweak from there with positive samples
(hopefully) in the cohort.

Length of fixation/processing/age of slides of the control vs cohort is
also something to think about.

yours,
Caroline



On Tue, Feb 21, 2023 at 12:53 PM Charles Riley via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> When performing IHC's for research projects is it recommended to use the
> same species for control tissues or will any tissues that the antibody has
> been validated for by the company be acceptable?
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Re: [Histonet] Super cold cryostat cutting head temperatures

[Caroline Miller via Histonet]

I have used down to -30 a few times for research samples, mainly to try and
get stuff to cut better rather than that is what was requested. Everything
gets very brittle at that temperature, including your hands after a short
while.

Never -60 though!

Maybe that is just for the peltier freezing mechanism? For putting the
tissue on the chucks with OCT glue?

I typically will set the specimen head a little lower than the chamber. But
only 3-4 degrees

This is the one I typically use and it is -40 max:
https://www.leicabiosystems.com/us/histology-equipment/cryostats/leica-cm3050-s/

I would be interested to know if anyone works at -60 in the LM world!

Caroline


On Thu, May 19, 2022 at 12:37 PM Colleen Forster via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Thank you Paul.
>
> Colleen
>
> On Thursday, May 19, 2022, P Sicurello  wrote:
>
> > Cryo-electron microscopy cuts thin sections at temperatures colder than
> > that.  It is used to preserve antigenicity in certain situations.
> >
> > However, a cryostat is not used.  An ultramicrotome fitted with specific
> > cryo attachments is used.
> >
> > Sincerely,
> >
> > Paula Sicurello
> >
> > Sent from my iPhone
> >
> > > On May 18, 2022, at 12:55 PM, Colleen Forster via Histonet <
> > histonet@lists.utsouthwestern.edu> wrote:
> > >
> > > FollowingI am in research and I have never cut this cold.
> > >
> > > Colleen Forster
> > >
> > >> On Wed, May 18, 2022 at 2:04 PM Ken Marzinsky via Histonet <
> > >> histonet@lists.utsouthwestern.edu> wrote:
> > >>
> > >> Can anyone tell me why research labs often require cryostat cutting
> head
> > >> temperatures down to -60C?
> > >>
> > >> Ken
> > >> Durham, NC
> > >> ___
> > >> Histonet mailing list
> > >> Histonet@lists.utsouthwestern.edu
> > >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> > >>
> > >
> > >
> > > --
> > > Colleen Forster HT(ASCP)QIHC
> > > BLS Histology and IHC Laboratory
> > > Jackson Hall, Room 2-155
> > > 321 Church St. SE
> > > Minneapolis, MN 55455
> > > 612-626-1930
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>
>
> --
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> BLS Histology and IHC Laboratory
> Jackson Hall, Room 2-155
> 321 Church St. SE
> Minneapolis, MN 55455
> 612-626-1930
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Re: [Histonet] Inconsistent H & E Staining

[Caroline Miller via Histonet]

Have you checked your dewaxing solutions? I have sometimes seen this when
slides are not fully dewaxed.

yours,
mills
ᐧ

On Wed, Nov 14, 2018 at 12:34 PM MONICA D. LOCKHART via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello Histo Fam!!
>
> Please help!
>
> My facility is experiencing frequent H & E staining problems.  Everything
> from cloudy cells on routine GI to "The Blank Look" on PNB's.
>
> We have made changes in some of the obvious areas like cassette whole size
> and discontinued the use of sponges at grossing.  However, with our
> biopsies, we still see varying results in our H staining.
>
> Though this is not an exhaustive list of all the things we have
> encountered or the number of changes we have made, hopefully this will give
> you an idea of what we are seeing and maybe you have ran across this
> problem and can share your wisdom.
>
> Any comments would be appreciated and most welcome.
>
>
> Monica D. Lockhart, BBA, HT (ASCP) PBT
> Supervisor Clinical Labs Histology
> Loyola University Medical Center
> 2160 S. First Ave, Bldg 110 Rm 2290
> Maywood, IL  60153
> (o) 708.327.2608
> (c) 708.692.8361
> monica.lockh...@luhs.org
>
>
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415 2187297
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Re: [Histonet] Oil Red O

[Caroline Miller via Histonet]

Hi Reuel,

In my experience this is the ORO crystallizing out on the section. I would
first filter your staining solution, then keep your staining times to as
short as you can. Also changing between 37 and room temperatures may be
useful too.

mills
ᐧ

On Wed, Oct 3, 2018 at 8:39 AM Reuel Cornelia via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I was wondering if you could help me know why our Oil Red O have some
> black snowflakes on our fat tissue hours after staining or after 24 hours
> more snowlflakes precipitation occurs.  The staining was reference was from
> Lillie RD, Ashburn. Please note that our staining works well between two to
> three hours. Is there a reason fro this precipitation?
>
>
> Reuel
>
> TSRH
>
> Dallas, TX
>
> --
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Re: [Histonet] Iron Stain

[Caroline Miller via Histonet]

HI Tasha,

The nuclear fast red will stain the nuclei red, the biebrich scarlet is a
connective tissue stain and may cover up the (sometimes sparse) prussian
blue localization of iron in the tissues. I, personally, would not consider
these reagents swappable.

In my experience nuclear fast red does last a long time, but I am also not
in a lab where expiration dates matter.

mills
ᐧ

On Wed, Aug 8, 2018 at 4:52 AM Campbell, Tasha M. via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Is nuclear fast red the only counter stain for Prussian blue stain?  I
> have a Masson's trichrome kit and was wondering if the Bierbech Scarlet
> could be a counterstain.  I won't be doing the iron stain very often at all
> so I am trying to keep dry reagents on hand to make up as needed so they do
> not expire so quickly.  Thanks!
>
>
>
>
> Tasha Campbell, B.S.,HTL(ASCP)
> Frederick Gastroenterology Associates
> 310 W. 9th St.
> Frederick, MD 21701
> 301-695-6800 ext. 144
>
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Director of Biology

www.3Scan.com
415 2187297
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Re: [Histonet] Iron Stain

[Caroline Miller via Histonet]

Oops, yes, I am mixing up neutral red - and the recipe from Bryan is what I
use - it last forever.

I have had luck with nuclear fast red from vector - comes made up. That
lasts a little longer than lab made.

mills
ᐧ

On Wed, Aug 8, 2018 at 12:52 PM Caroline Miller  wrote:

> HI Tasha,
>
> The nuclear fast red will stain the nuclei red, the biebrich scarlet is a
> connective tissue stain and may cover up the (sometimes sparse) prussian
> blue localization of iron in the tissues. I, personally, would not consider
> these reagents swappable.
>
> In my experience nuclear fast red does last a long time, but I am also not
> in a lab where expiration dates matter.
>
> mills
> ᐧ
>
> On Wed, Aug 8, 2018 at 4:52 AM Campbell, Tasha M. via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
>> Is nuclear fast red the only counter stain for Prussian blue stain?  I
>> have a Masson's trichrome kit and was wondering if the Bierbech Scarlet
>> could be a counterstain.  I won't be doing the iron stain very often at all
>> so I am trying to keep dry reagents on hand to make up as needed so they do
>> not expire so quickly.  Thanks!
>>
>>
>>
>>
>> Tasha Campbell, B.S.,HTL(ASCP)
>> Frederick Gastroenterology Associates
>> 310 W. 9th St.
>> Frederick, MD 21701
>> 301-695-6800 ext. 144
>>
>> ___
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>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
>
> --
> Caroline Miller (mills)
> Director of Biology
>
> www.3Scan.com
> 415 2187297
>
>
>

-- 
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Director of Biology

www.3Scan.com
415 2187297
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Re: [Histonet] GI biopsies

[Caroline Miller via Histonet]

In London, at the Hammersmith, we used to do 3 on two slides, one H+E and
the other spare.

mills
ᐧ

On Mon, Jun 4, 2018 at 11:57 AM Sousa, Katie via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Taking a survey: how many levels do you all cut for GI biopsy specimens?
>
> Katie Sousa
> Supervisor Histology
> Baystate Medical Center
> 361 Whitney Ave., Holyoke, MA 01040
> Telephone: 413-322-4786  Fax: 413-322-4790
> katie.so...@baystatehealth.org
>
>
> --
> Please view our annual report at http://www.bhannualreport.org
>
>
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Re: [Histonet] tissue banks

[Caroline Miller via Histonet]

Thanks for the tip!

mills

On Tue, Apr 3, 2018 at 2:39 PM, Adrienne Anderson <rennie1...@yahoo.com>
wrote:

> Hi Caroline and Cassie,
>
> I used to be the histology manager for Folio Biosciences (now called Folio
> Conversant I believe). It's a smaller company based out of Columbus, OH,
> and they are great to work with!
>
> Let me know if you have any questions about them.
>
> Best regards,
> Adrienne Anderson
>
> On Monday, April 2, 2018, 2:25:46 PM MDT, Caroline Miller via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
>
> I am very interested in this also. We have found a few mainly from this
> google search:
> https://www.google.com/search?ei=WYXCWt_bKoKh0wLn0rDYCQ=
> human+tissues+for+research=human+tissues+for+research_
> l=psy-ab.3...0.0.0.141303.0.0.0.0.0.0.0.0..0.00...1..64.
> psy-ab..0.0.00.XTGjP8zFu9Y
>
> We have tried cureline and didn't have too much success.
>
> http://ndriresource.org/ looks interesting but haven't tried it yet
>
> and this one from the NCI looks the most legit, the application procedure
> seems quite straight forward, but we haven't tried it yet:
> https://www.chtn.org/
>
> Where we have had success is through organ procurement organizations, where
> you have to submit an application for review, and you are second on the
> list after the donor network. This may be a little too involved for your
> process / requirements though:
> IIAM.org
> DNW.org
>
> Happy to hear of other experiences though so please forward!
>
> yours,
> mills
>
> On Mon, Apr 2, 2018 at 11:32 AM, Cassie P. Davis via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > Good afternoon Histology Folks,
> >
> >10 or more years ago there used to be a couple of companies
> > that would buy body fluids and tissues from labs that were ready to be
> > disposed of. Do they still exist and would you please share their contact
> > information?
> >
> >
> > thanks,
> >
> > Cassandra Davis
> > Histology Technician
> > AP Laboratory
> > 302-575-8095
> > Email:  cda...@che-east.org<mailto:cda...@che-east.org>
> >
> >
> >
> > Confidentiality Notice:
> > This e-mail, including any attachments is the property of Trinity Health
> > and is intended for the sole use of the intended recipient(s). It may
> > contain information that is privileged and confidential.  Any
> unauthorized
> > review, use, disclosure, or distribution is prohibited. If you are not
> the
> > intended recipient, please delete this message, and reply to the sender
> > regarding the error in a separate email.
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> >
>
>
>
> --
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> Director of Histology
> 3Scan.com
> 415 2187297
>
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Re: [Histonet] tissue banks

[Caroline Miller via Histonet]

I am very interested in this also. We have found a few mainly from this
google search:
https://www.google.com/search?ei=WYXCWt_bKoKh0wLn0rDYCQ=human+tissues+for+research=human+tissues+for+research_l=psy-ab.3...0.0.0.141303.0.0.0.0.0.0.0.0..0.00...1..64.psy-ab..0.0.00.XTGjP8zFu9Y

We have tried cureline and didn't have too much success.

http://ndriresource.org/ looks interesting but haven't tried it yet

and this one from the NCI looks the most legit, the application procedure
seems quite straight forward, but we haven't tried it yet:
https://www.chtn.org/

Where we have had success is through organ procurement organizations, where
you have to submit an application for review, and you are second on the
list after the donor network. This may be a little too involved for your
process / requirements though:
IIAM.org
DNW.org

Happy to hear of other experiences though so please forward!

yours,
mills

On Mon, Apr 2, 2018 at 11:32 AM, Cassie P. Davis via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Good afternoon Histology Folks,
>
> 10 or more years ago there used to be a couple of companies
> that would buy body fluids and tissues from labs that were ready to be
> disposed of. Do they still exist and would you please share their contact
> information?
>
>
> thanks,
>
> Cassandra Davis
> Histology Technician
> AP Laboratory
> 302-575-8095
> Email:  cda...@che-east.org
>
>
>
> Confidentiality Notice:
> This e-mail, including any attachments is the property of Trinity Health
> and is intended for the sole use of the intended recipient(s). It may
> contain information that is privileged and confidential.  Any unauthorized
> review, use, disclosure, or distribution is prohibited. If you are not the
> intended recipient, please delete this message, and reply to the sender
> regarding the error in a separate email.
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Re: [Histonet] DAKO

[Caroline Miller via Histonet]

I have issues with the fact that the website no longer has datasheets
available and you have to email the customer services to get them!!

It has been like that for almost a year, even though they say they are
working on it.

yours,
mills

On Thu, Feb 8, 2018 at 5:12 AM, Lisa Parsons via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Has anyone had any issues with their service/contracts from DAKO now that
> they are just Agilent?  My new contract is very disappointing.
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Re: [Histonet] Elastic Stain

[Caroline Miller via Histonet]

In the UK clinical lab I started in we used Miller's elastin, which I think
was a resourcinol formulation (digging from 20 years ago in my brain). I
tried to find that in the US but never did. I loved that stain, worked well
every time! We would counterstain with a van-Giesen, looked stunning! the
elastin came out very dark blue / black.

mills

On Wed, Nov 8, 2017 at 6:31 AM, Terri Braud via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> We love the Aldehyde Fuchsin with a fast-green counterstain.  Once the
> initial working stain is made up, it is a fast and easy stain to perform,
> not to mention just pretty - purple fibers against a green background.
>
> Terri L. Braud, HT(ASCP)
> Anatomic Pathology Supervisor
> Laboratory
> Holy Redeemer Hospital
> 1648 Huntingdon Pike
> Meadowbrook, PA 19046
> ph: 215-938-3689
> fax: 215-938-3874
> Care, Comfort, and Heal
> *
>
>
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Re: [Histonet] Metal embedding molds-large

[Caroline Miller via Histonet]

hey, we do all our embedding in those molds, and here is what I suggest:

1 - Make sure to have enough wax in the back of the molds, all the way
until it is on the lip of the cassette - you may need to refill a  bunch of
times because the wax drains out (why regular sakura cassettes do not fit
in these molds, also made by sakura I really don't know). But you need to
wait until it hardens enough, but not too much to leave a transition
between the two fill waxes. This will prevent the cassette from coming away
when you pop it out
2 - Make sure the block is nice and cold, wait 10 minutes longer than you
think you have to
3 - Use a spatula or other strong item to place under the label side of the
cassette and pop out of the mold, if you get any resistance then WAIT some
more! (again them being cold is really important)

good luck, happy to answer any clarifying questions!

mills

On Wed, Nov 8, 2017 at 12:30 PM, Bryan Llewellyn via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Sorry!
>
> That should be TSP - trisodium phosphate - not TCP, which might make it
> worse.
>
> Bryan
>
>
>
> Bryan Llewellyn wrote:
>
>> This used to be a common problem years ago. It is due to crud buildup on
>> the metal. Boil them with TCP for half an hour, then thoroughly wash
>> them in cold water. Coat them with a VERY light smear of glycerol before
>> you use them, preferably each time. That should help.
>>
>> Bryan Llewellyn.
>>
>> Diane Satterfield via Histonet wrote:
>>
>>> We are using large metal molds to embed mouse brains.  We are having a
>>> hard time getting to block out of the molds, the paraffin blocks are
>>> sticking.  Sometimes they are coming out cracked.  Sometimes the
>>> cassette comes off the paraffin block.  Any idea why this is
>>> happening? Any advice on how to fix this problem?
>>>
>>>
>>> Diane L. Satterfield, BS
>>> Manager Brain Tumor BioRepository
>>> Research Program Leader
>>> Duke University Medical Center
>>> Brain Tumor Center Biorepository and Database
>>>
>>> diane.satterfi...@duke.edu
>>> office  919-684-4642
>>> pager  919-970-7328
>>> fax  919-684-4975
>>>
>>> CONFIDENTIALITY NOTICE:  The information contained in this electronic
>>> mail is sensitive, protected information intended only for the
>>> addressee(s).  Any other person, including anyone who believes he/she
>>> might have received it due to an addressing error, is requested to
>>> notify the sender immediately by return electronic mail, and to delete
>>> it without further reading or retention.  The information is not to be
>>> forwarded to or shared unless in compliance with Duke Medicine
>>> policies on confidentiality and/or with the approval of the sender.
>>>
>>>
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>>>
>>
>
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Re: [Histonet] Type of Nitrile Gloves in your lab

[Caroline Miller via Histonet]

I love these:
*https://www.net32.com/ec/cobalt-nitrile-exam-gloves-xsmall-100-powderfree-d-135964
*

*This is also the cheapest place I have found them listed as well. *

*yours,*
*mills*

On Tue, Nov 7, 2017 at 8:21 AM, Haley Huggins via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I like the Halyard brand.  We also use Confiderm from McKesson, those are
> pretty good too.
>
> *Haley Huggins, HT (ASCP)cm*
> *Technical Lab Supervisor*
> *1050 Las Tablas Rd, Suite 14*
> *Templeton, CA 93465*
> *Office: 877-230-1518*
>
> On Mon, Nov 6, 2017 at 10:25 AM, Cooper, Brian via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > Hi,
> >
> > Does anyone out there use a brand of nitrile glove that they really like?
> > The ones we use here are not so good.  When we pull them on, the part
> > closet to the wrist often tears off, leaving behind a nice blue rubber
> > band.  At least several gloves per box have tears in them before we even
> > put them on.  One of our Quality Managers asked us to come up with
> > alternatives.  Can you help me out?   You can email me offline if you
> don't
> > want to name particular brands for everyone to see.  PLEASE HELP!!!
> >
> > Thanks,
> >
> > Brian D. Cooper, HT (ASCP)CM | Histology Supervisor
> > Department of Pathology and Laboratory Medicine
> > Children's Hospital Los Angeles
> > 4650 Sunset Blvd MS#43- Los Angeles, CA 90027
> > Ph: 323.361.3357 Pager: 213-209-0184
> > bcoo...@chla.usc.edu
> >
> >
> > CONFIDENTIALITY NOTICE: This e-mail message, including any attachments,
> > is for the sole use of the intended recipient(s) and may contain
> > confidential
> > or legally privileged information. Any unauthorized review, use,
> disclosure
> > or distribution is prohibited. If you are not the intended recipient,
> > please
> > contact the sender by reply e-mail and destroy all copies of this
> original
> > message.
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Re: [Histonet] UV light for von Kossa

[Caroline Miller via Histonet]

Yes, I have used desk lamps / foil and also the window on a sunny day!
(caveat - mine was all research and I am unsure of the Clia rules in this
space)

yours,
mills

On Mon, Jul 31, 2017 at 7:06 AM, Mayer,Toysha N via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Michael,
>
> We just use one of the small desk lamps at a cutting station, the back of
> the embedder, and foil to reflect.  It works great.  Just place the coplin
> jar with the slides on top of the control panel of the embedder.  Adjust
> the lamp to the top of the jar and wrap it in foil.  The heat penetrates
> and is reflected. Voila, a von Kossa.
> Toysha
>
>
>
> Message: 3
> Date: Fri, 28 Jul 2017 14:25:01 -0400
> From: Michael Kent 
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] UV light for von Kossa
> Message-ID: <2c562a902723b33a4e73859c01ab3...@mail.gmail.com>
> Content-Type: text/plain; charset="UTF-8"
>
> Hi Histonet,
>
> I am looking for a cost-effective UV light for von Kossa staining.  The
> clouds are getting tin the way.
>
> Thank you, and have a god weekend.
>
> Mike
>
>
> --
>
> Subject: Digest Footer
>
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> --
>
> End of Histonet Digest, Vol 164, Issue 25
> *
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> confidential, and/or protected from disclosure. This e-mail message may
> contain protected health information (PHI); dissemination of PHI should
> comply with applicable federal and state laws. If you are not the intended
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> further review, disclosure, use, dissemination, distribution, or copying of
> this message or any attachment (or the information contained therein) is
> strictly prohibited. If you think that you have received this e-mail
> message in error, please notify the sender by return e-mail and delete all
> references to it and its contents from your systems.
>
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Re: [Histonet] What happened to Ultraclone/PGP9.5?

[Caroline Miller via Histonet]

I have had very good luck with the Dako pgp9.5 in both slides, as well as
whole mount staining on skin.

mills

On Sun, Jun 4, 2017 at 3:48 PM, Eddie Martin via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Novocastra has been selling pgp 9.5 for years. I've used it on frozen
> sections and FFPR on 3 microns as well as 50 micron sections and it stains
> very well.
>
> Sent from my iPhone
>
> > On Jun 3, 2017, at 3:22 PM, Hobbs, Carl  wrote:
> >
> >
> >
> > Ultraclone
> > THE supplier of PGP9.5 for many years !
> > Respect to them/him/her/LTB
> > No longersigh.
> > Ultraclone:  "legendary"...chuckle
> >
> >
> > Sureother Suppliers now offer anti PGP9.5 abs
> > Why then do they still call it "PGP9.5"??
> > We now know what the protein is.
> >
> > PGP: "Pretty good protein" I recall?
> > Why the 9.5?
> > Usually a clone but, but Ultraclone ab was a rabbit poly.
> >
> > One could only order via fax, I recall.
> > Isle of Wight location: Rossiters Farm.
> >
> > Any more  info re Ultraclone's history  would be most appreciated.
> >
> > Curiously
> >
> > Carl
> >
> >
> >
>
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Re: [Histonet] CD8 for rat - also happy to take human suggestions

[Caroline Miller via Histonet]

Thank you for all the suggestions Histonet, you wonders of knowledge!

Happy to share if anyone wants a collated list, let me know

Happy Friday!

mills

On Thu, Apr 20, 2017 at 12:13 PM, Caroline Miller  wrote:

> Hi Histonet!
>
> I am on the search for a good CD8 antibody, I have done a literature dive
> and cannot find a consistently used one. I currently have abcam ab131500 Rb
> (monoclonal) and it is just not working at all.
>
> Does anyone have any advice in this space. I am totally interested in
> clinical human antibodies too as they often have cross reactivity.
>
> Any other nuances in the staining that people have found would also be
> great.
>
> thanks folks!
>
> mills
>
> --
> Caroline Miller (mills)
> Director of Histology
> 3Scan.com
> 415 2187297 <(415)%20218-7297>
>



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[Histonet] CD8 for rat - also happy to take human suggestions

[Caroline Miller via Histonet]

Hi Histonet!

I am on the search for a good CD8 antibody, I have done a literature dive
and cannot find a consistently used one. I currently have abcam ab131500 Rb
(monoclonal) and it is just not working at all.

Does anyone have any advice in this space. I am totally interested in
clinical human antibodies too as they often have cross reactivity.

Any other nuances in the staining that people have found would also be
great.

thanks folks!

mills

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Re: [Histonet] processor died overnight

[Caroline Miller via Histonet]

Yes, totally +1 to Rene, they should be fine. 
(That has totally happened to me too)!

Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

> On Apr 19, 2017, at 6:41 AM, Rene J Buesa via Histonet 
>  wrote:
> 
> In 100% EthOL the tissues are completely "salvaged" and you can prepare the 
> program to continue the steps until melted paraffin.If there are delicate 
> tissue perhaps they will be "over-dried" but that is easily "compensated" 
> during microtomy.René 
> 
>On Wednesday, April 19, 2017 9:01 AM, Lauren Sweeney via Histonet 
>  wrote:
> 
> 
> Hello Histoworld,
> 
> I came in this morning to find that the processor died halfway through 
> process last night. The tissues are in 100% ETOH exactly half point. We do 
> have a back- up processor. In your professional experiences, would these 
> tissues be salvageable? Could I create a new program on the backup processor 
> that finishes the process from that point and transfer the tissues over?
> 
> Thanks.
> 
> 
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> 
> 
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Re: [Histonet] help!!

[Caroline Miller via Histonet]

Blanca,
Here are my feelings on this, and I am sure a lot of other folks have feels
here too, so please chime in.

1 - I feel that most clinical labs are more on the IHC bandwagon and
research labs are IF (with the exception of IgG staining in kidney biopsies
or bullous disease in skin- which is because the antibodies don't like
formalin fixing (if this is now wrong I am sorry, I haven't been in a
clinical lab in quite a while). Research labs are often also working with
genetically encoded fluorophores such as GFP, YFP, mCherry
2 - Formalin fixation (especially over fixation) can often lead to a large
amount of autofluorescence in the 488 region, which is a common place for
secondary antibodies and also GFP. Research labs have a lot more control
over their fixation protocols.
3 - The microscopes commonly available to clinical labs are bright field
scopes and in research labs fluorescent scopes
4 - Fluorescence can provide more contrast to a positively localized
fluorophore, but sometimes at the detriment of viewing the overall
morphology of the tissue like you get with bright field IHC and a nuclear
counterstain.
5 - Research lab protocols are often very 'experimental' and can lead to
increased tissue damage, which again is not viewed under the fluorescence
microscope (as much). Clinical labs have lots of experience and also
defined protocols that work well in the IHC / bright field space.
6 - the only real difference is the detection method, you can use any
primary antibody with either ABC/ impress / enzyme based methods or with
fluorophore conjugated secondaries.

So, in short - no *real* reason, but mainly that is the way things shook
out.

I could go on about researchers not understanding how to take photos on a
bright field scopes too, but that is too broad a statement, but as a core
director I saw them being more comfortable with the fluorescent methods :)

mills




On Thu, Apr 13, 2017 at 6:09 AM, Blanca Lopez via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello!
> I just need a help with a simple question...Is anyone can explain me what
> is the purpose between performing immunohistochemistry and
> Immunofluorescence?
> Thanks  :)
>
> Blanca Lopez
> Histotech (ASCP)
> UTSW Tissue Resource K1.210
> Simmons Comprehensive Cancer Center
> UT Southwestern Medical Center
> Telephone: 214-648-7598
> Email: blanca.lo...@utsouthwestern.edu
>
>
> 
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>
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Re: [Histonet] No Ribbon

[Caroline Miller via Histonet]

Point taken, thanks Elizabeth.

My clinical days are long gone, but that is an important consideration.

yours,
mills

On Tue, Apr 11, 2017 at 11:13 AM, Elizabeth Chlipala <l...@premierlab.com>
wrote:

> I might get some individuals upset but we really need to watch what we do
> in the histology lab and how that effects the precision of our process.
> When you breath on the block you are basically generating a thicker
> section, it's not a good practice to keep.  Trust me I used to do this in
> the past.  Thicker sections especially for IHC will generate a more intense
> stain, most individuals may think that that should not be an issue but it
> is - in the clinical setting especially when you are staining for
> therapeutic markers such as Her2 and PD-L1 - 510K cleared kits for these
> recommend specific section thickness settings and in the research setting
> if you are running any type of analysis on a batch of samples.  If your
> microtome is set at 4 microns and you are breathing on the block you are
> not cutting at 4 microns, you are cutting thicker than that and a thicker
> section will increase staining intensity and therefore alter the results.
>
> Just my two cents.
>
> 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
>
>
> -Original Message-
> From: Caroline Miller via Histonet [mailto:histonet@lists.
> utsouthwestern.edu]
> Sent: Tuesday, April 11, 2017 11:59 AM
> To: Paula Keene Pierce
> Cc: Histonet Histonet
> Subject: Re: [Histonet] No Ribbon
>
> Yes, +1 to Paula, also:
> - I often found they are too sharp (!!) to ribbon so a gentle wipe (in the
> direction of the bevel) with a kimwipe helps
> - Also 'huffing' slightly on the block will warm the paraffin and both
> smooth out the wrinkles as well as make the sections stick together
>
> :)
>
> mills
>
> On Tue, Apr 11, 2017 at 10:37 AM, Paula Keene Pierce via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > Hi,
> > the blades are packed with a tiny, bit of oil. This can cause you to
> > not obtain a ribbon.
> > Try gently cleaning with xylene, followed by alcohol before
> > sectioning. Paula Keene Pierce, BS, HTL(ASCP)HTPresidentExcalibur
> > Pathology, Inc.5830 N Blue Lake DriveNorman, OK 73069PH
> > 405-759-3953FAX 405-759-7513www.excaliburpathology.com
> >
> >   From: Gareth Davis via Histonet
> > <histonet@lists.utsouthwestern.edu>
> >  To: "Histonet@lists.utsouthwestern.edu" <histonet@lists.
> > utsouthwestern.edu>
> >  Sent: Tuesday, April 11, 2017 11:55 AM
> >  Subject: [Histonet] No Ribbon
> >
> > I feel like I should be able to figure this one out, but I have been
> > having trouble with getting a ribbon for a while now.  I have a Leica
> > Microtome
> > RM2125 and I use either a AccuEdge blade (I get one block to cut then
> > it starts to compress) or a StatCut blade (sections do not stick
> > together at all), both High Profile.  The angle is about 4 microns.  I
> > change the angle a lot to see if that makes a difference and it
> > doesn't.  There are so many factors that could be the issue, I think
> > lack of humidity may be the main one. So, suggestions please!!
> >
> > --
> > Ms. Gareth B. Davis, B.S., HT, QIHC (ASCP)cm Yuma Gastroenterology
> > Yuma, AZ 85364
> > 928-248-5259
> > ___
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> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
> >
> >
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> >
>
>
>
> --
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> Director of Histology
> 3Scan.com
> 415 2187297
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Re: [Histonet] No Ribbon

[Caroline Miller via Histonet]

Yes, +1 to Paula, also:
- I often found they are too sharp (!!) to ribbon so a gentle wipe (in the
direction of the bevel) with a kimwipe helps
- Also 'huffing' slightly on the block will warm the paraffin and both
smooth out the wrinkles as well as make the sections stick together

:)

mills

On Tue, Apr 11, 2017 at 10:37 AM, Paula Keene Pierce via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi,
> the blades are packed with a tiny, bit of oil. This can cause you to not
> obtain a ribbon.
> Try gently cleaning with xylene, followed by alcohol before
> sectioning. Paula Keene Pierce, BS, HTL(ASCP)HTPresidentExcalibur
> Pathology, Inc.5830 N Blue Lake DriveNorman, OK 73069PH 405-759-3953FAX
> 405-759-7513www.excaliburpathology.com
>
>   From: Gareth Davis via Histonet 
>  To: "Histonet@lists.utsouthwestern.edu"  utsouthwestern.edu>
>  Sent: Tuesday, April 11, 2017 11:55 AM
>  Subject: [Histonet] No Ribbon
>
> I feel like I should be able to figure this one out, but I have been having
> trouble with getting a ribbon for a while now.  I have a Leica Microtome
> RM2125 and I use either a AccuEdge blade (I get one block to cut then it
> starts to compress) or a StatCut blade (sections do not stick together at
> all), both High Profile.  The angle is about 4 microns.  I change the angle
> a lot to see if that makes a difference and it doesn't.  There are so many
> factors that could be the issue, I think lack of humidity may be the main
> one. So, suggestions please!!
>
> --
> Ms. Gareth B. Davis, B.S., HT, QIHC (ASCP)cm
> Yuma Gastroenterology
> Yuma, AZ 85364
> 928-248-5259
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>
>
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Re: [Histonet] TUNEL

[Caroline Miller via Histonet]

I have done that a lot and I don't see why it would not pick up hx or DAPI.
Was there anything left on the slide? Maybe it was mounted in aqueous
already??

mills

On Fri, Mar 3, 2017 at 11:42 AM, Bernice Frederick via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello all.
> Would there be any reason that slides stained with TUNEL (ISH) would not
> pick up the Mayers counterstain? We use it for IHC and it is fine. Mayers
> for 7 minutes and then ammonia water for 1 minute. We do that rather than
> rinse for 15 minutes and there are usually no issues. Could a reagent in
> the stain be causing it? DAPI is used...
> Bernice
>
> Bernice Frederick HTL (ASCP)
> Senior Research Tech
> Pathology Core Facility
> Robert. H. Lurie Cancer Center
> Northwestern University
> 710 N Fairbanks Court
> Olson 8-421
> Chicago,IL 60611
> 312-503-3723
> b-freder...@northwestern.edu
>
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Re: [Histonet] hand staining immunos

[Caroline Miller via Histonet]

Agreed! Don't believe the 'single use only' on the coverplates - wash and
reuse them. I use tube racks to stand them up to dry, then put the back in
the original packaging. after a few years some of them might lose their
spring and you will need to replace some at that point.

yours,
mills

On Mon, Feb 13, 2017 at 7:42 AM, Paula Keene Pierce <
pa...@excaliburpathology.com> wrote:

> https://www.fishersci.com/shop/products/7219950/7219950#?keyword=sequenza
>
> These are the cover plates.
>
> I have no financial interest in Thermo Fisher, but have used the system
> for years.
>
>
>
>
> Paula Keene Pierce, BS, HTL(ASCP)HT
> President
> Excalibur Pathology, Inc.
> 5830 N Blue Lake Drive
> Norman, OK 73069
> PH 405-759-3953 <(405)%20759-3953>
> FAX 405-759-7513 <(405)%20759-7513>
> www.excaliburpathology.com
>
>
> --
> *From:* Caroline Miller via Histonet <histonet@lists.utsouthwestern.edu>
> *To:* Ranna Mehta <ranna0...@gmail.com>
> *Cc:* "Histonet@Lists. Edu" <histonet@lists.utsouthwestern.edu>
> *Sent:* Monday, February 13, 2017 9:06 AM
> *Subject:* Re: [Histonet] hand staining immunos
>
> This is a great system for semi-automated immuno. I find the results a
> little cleaner than performing the technique straight on the slide. No need
> to buy the whole set up (it is just a glorified timer)- just the racks and
> clips are enough!
>
> https://www.fishersci.com/shop/products/thermo-
> scientific-shandon-sequenza-immunostaining-center-2/p-4010433
>
> mills
>
> On Thu, Feb 9, 2017 at 11:32 AM, Ranna Mehta via Histonet <
> histonet@lists.utsouthwestern.edu> wrote:
>
> > Hi Jennifer,
> >
> >  How many slides are you planning to stain in routine?  Once you will
> > understand the basic, it will be a very interesting field. If you like, I
> > can help you to walk through process with step by step. send me a message
> > through my email.All the best and thanks.
> >
> > Regards
> > Ranna
> >
> > On Thu, Feb 9, 2017 at 12:00 PM, <histonet-request@lists.
> > utsouthwestern.edu>
> > wrote:
> >
> > > Send Histonet mailing list submissions to
> > >histonet@lists.utsouthwestern.edu
> > >
> > > To subscribe or unsubscribe via the World Wide Web, visit
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> > >1. Looking for a Histology Workshop (Jessica Riggleman)
> > >2. Re: Looking for a Histology Workshop (Jessica Riggleman)
> > >3. hand staining immunos (Jennifer)
> > >4. Re: hand staining immunos (Rene J Buesa)
> > >5. Re: hand staining immunos (Walter Benton)
> > >
> > >
> > > --
> > >
> > > Message: 1
> > > Date: Wed, 8 Feb 2017 18:07:24 +
> > > From: Jessica Riggleman <jriggle...@globusmedical.com>
> > > To: "histonet@lists.utsouthwestern.edu"
> > ><histonet@lists.utsouthwestern.edu>
> > > Subject: [Histonet] Looking for a Histology Workshop
> > > Message-ID:
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> >
> > > Content-Type: text/plain; charset=UTF-8
> > >
> > >
> > > Hi Everyone,
> > >
> > > I am looking for workshop or class for Histology/Histotechnology that
> > > lasts anywhere from 1 week to 3 months in the U.S.
> > > Preferably in-person and not online.
> > >
> > > If you have any suggestions or contacts, please let me know.
> > >
> > > Thank You,
> > > Jessica
> > >
> > > _
> > >
> > > Jessica Riggleman | Research Associate
> > >
> > > Globus Medical, Inc.
> > > Valley Forge Business Center
> > > 2560 General Armistead Avenue | Audubon, PA 19403
> > > Ph: (610) 930-1800 ext. 2583 <(610)%20930-1800> | Fax:
> > > website: http://www.globusmedical.com | e

Re: [Histonet] grad student problem

[Caroline Miller via Histonet]

I hate to say it but I think these tissues are toast. It seems they had bad
fixation or handling to start with, and I would question whether the
tissues were left out to dry before fixation or freezing. Or whether they
got fixed and then sucrose infiltrated (which is my method of choice if the
antibody allows)

Also - what type of tissues? If it is lymph nodes then maybe there is too
much fatty tissue and that is why it is not sectioning.

I have had a hard time with T-cell markers in paraffin so I do see the
hesitation in paraffin embedding. If you do go that route then certainly
thaw in formalin, not straight water.

Not much help, sorry! But sometimes it is just better to go back and plan
the experiment properly in the first place than trying to mess around with
old bits of tissue - because even if you could get it on the slide, would
you really trust the results of the immunostaining???

Happy New Year All!

yours,
mills

On Tue, Jan 3, 2017 at 10:05 AM, Roberta Horner via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I got the following from a grad student here at Penn State. I am not sure
> how to solve his problem if possible. Does anyone have any suggestions I
> can forward?
> Roberta Horner
> Animal Diagnostic Lab
> Penn State University
>
> "I am having some difficulties sectioning mouse tumor samples for
> immunofluorescent analysis.  We originally went the OCT route because we
> are staining for T cell markers and were worried that the heating that
> occurs during paraffin embedding would compromise the T cell receptor.  The
> samples are a little old, but we are hoping to section and stain for immune
> cell infiltrates.  When sectioning with the cryostat, the tissue and OCT is
> quite brittle and the sample is not intact enough to transfer to a slide.
> Two colleagues have given the following suggestions:
> 1. Thaw the OCT blocks, remove the tissue, and place in a new block with
> fresh OCT media.  I've tried this technique on a few practice samples and
> the new OCT media seems to be less brittle and I'm able to get tissue on a
> slide, however the tissue itself still seems to be poor with either freeze
> or sectioning artifacts.
> 2. Thaw the OCT blocks in water, remove the tissue and place in formalin
> overnight, place in 70% EtOH, then paraffin embed.  Section on a
> microtome.  Check the fluorescently labelled antibody data sheet to see if
> paraffin embedding interferes with binding.  Try to stain and see what
> happens.
>
> I was hesitant to try the second suggestion because I have found no
> protocol that takes tissue originally stored in OCT blocks and subsequently
> redirects them to formalin and paraffin for microtome sectioning.  If you
> have any recommendations on how to move forward and section these difficult
> samples, or know anyone at the diagnostics lab or at Penn State that could
> help, that would be much appreciated!"
>
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Re: [Histonet] Weigert's hematoxylin for IHC

[Caroline Miller via Histonet]

I would caution using Weigerts as a counterstain for IHC because it is
pretty dark in color and could be confused with the brown of the staining
you are looking for in your IHC. Have you tried Mayer's? That often
provides a pleasing stain with minimal effort.

You would counterstain after the DAB development steps, prior to mounting
the slides with coverslips.

I have never seen coral staining though, so this is only a guess from what
works in regular mammalian tissues, good luck!

yours,
mills

On Tue, Nov 22, 2016 at 2:24 PM, Esther C Peters via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Could someone advise me on whether Weigert's iron hematoxylin can be used
> as the counterstain for nuclei in IHC? We need to avoid using Harris's
> hematoxylin because that will stain mucocytes in the coral tissues we are
> working with. If it can be used, would that be after all antibody steps
> have been done? Thank you!
>
>
> Esther Peters
>
>
> Esther C. Peters, Ph.D.
> Term Associate Professor
> Environmental Science & Policy
> George Mason University
> 4400 University Drive, MS 5F2
> Fairfax, VA 22030-
> Office: David King Hall, Room 3050
> Phone: 703-993-3462
> Fax: 703-993-1066
> e-mail: epete...@gmu.edu
>  xC0ytBErXdaN3U3lGqWmZNdAI_N-4nsEb0IjgUpeIoQa7EcVMJMh2oePPP
> KrrDjhwOvk.=http%3a%2f%2fesp.gmu.edu>http://esp.gmu.edu
>
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Re: [Histonet] PGP 9.5 Ab x-reactivity

[Caroline Miller via Histonet]

I am interested in this too please! However I will be performing the stain
whole mount on skin and gut

Thanks in advance!

yours,
mills

On Tue, Nov 8, 2016 at 3:44 AM, Hobbs, Carl via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

>
> Hi
> I'd be grateful if anyone can confirm whether or not Dako's  Z511601-2   or
> Leica's ( Novacastra's) PGP9.5-L-CE  anti PGP 9.5 antibodies work on
> mouse/rat Formalin-fixed Paraffin wax sections.
>
> Thank you
>
>
> Carl
>
>
>
> Carl Hobbs FIBMS
> Histology and Imaging Manager
> Wolfson CARD
> Guys Campus, London Bridge
> Kings College London
> London
> SE1 1UL
>
> 020 7848 6813
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Re: [Histonet] Plant tissue

[Caroline Miller via Histonet]

I am interested in this too! Please forward to all. What are the best
resources for how to handle plant material for microscopic analysis?

thanks!
mills

On Fri, Oct 21, 2016 at 12:28 PM, John Shelley via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Happy Friday!
>
> I have given a request for service to me to do some plant tissue histology
> and I must admit this is a new area for me. I plan to do both paraffin and
> frozen sectioning and would like to ask if there may be anyone has a
> protocol on how to handle such tissue in regards to a tissue processing
> schedule and also steps to take for freezing. Any information would be
> greatly appreciated.
>
> Have a great weekend!
>
> Kind Regards!
>
> John J Shelley
> Histology Core Manager
> Sanford Burnham Prebys Medical Discovery Institute at Lake Nona
>
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Re: [Histonet] Substituting formalin by 70%ethanol in fixed samples.

[Caroline Miller via Histonet]

Hi Julio,

I can speak to a research / core setting where I often advised my users to
place their tissues in 70% ethanol following adequate formalin fixation to
maintain epitopes for potential downstream IHC studies. I have found tissue
can be placed in 70% and maintained indefinitely. They do not 'unfix'.

Recently though I was speaking to a pathologist who doesn't like that
protocol as she sees nuclear changes in tissues that have been in 70% for
extended periods.

So, yes, I think that idea is good in principle. I would say that the staff
time taken, and extra reagent costs, of changing all those biopsies over
into another solution, plus the additional chemical waste generated is not
worth simply making sure the original formalin containers are well sealed
after grossing  and / or purchasing one of those specimen collection
cupboards that filters the air coming through.

I do understand the dangers of formalin, but considering how much of it is
in use and how many of us on this list have been exposed to it for the
majority of our lives and we are still OK! (well, maybe, I suppose that
depends on your definition of OK is :)

Leica seem to think this is OK too!
http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-5-practical-procedures-to-optimise-quality-the-effects-of-heat-and-microwaves/

Ultimately you would have to perform studies to check the effects on your
downstream processes and proof this procedure in your own lab.

yours,
mills



On Sun, Oct 16, 2016 at 10:57 AM, Julio Benavides via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

>
> Hi there,
>
> This is me again with formalin issues. The health and safety officer of my
> institute keeps on in her crusade of eliminating formalin from the world.
> After your helpful emails, I convinced her that you do need formalin to fix
> samples, other approach would need to much setup IHC protocols.  Then and
> with good intention at heart, she has suggested that, one possible solution
> to reduce formalin presence in the institute could be, once samples have
> been fixed in buffered formalin (10%) and embedded in wax, to substitute it
> by 70% ethanol until the sample is discharged. Has anybody tried anything
> similar? What do you think? Would it be possible to come back to formalin
> in case of necessity (let´s say we want to retrim fixed samples after they
> have been in ethanol 70% for 5 months… would you trust them for IHC? HE?).
>
> Thanks again for your help
>
> Julio
>
>
>
> ___
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Re: [Histonet] Vacuolated and torn?

[Caroline Miller via Histonet]

If it is a block issue you can also often see 'white' bits on the block
surface that correlate to the torn areas on the slide, try 'polishing' the
block at 1um until they go away, cool and then try sectioning again.

I am unsure how that relates to vacuolation - as I have usually seen that
referred to an intracellular event - and not to refer to a regular hole.

I also suggest having someone else cut a block and then compare.

yours,
mills

On Thu, Oct 6, 2016 at 3:43 PM, Cartun, Richard via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Microtomy is so important, but often over-looked.  You might try giving
> the paraffin block(s) to different people and have them all cut H, and
> then have your pathologist compare them.  If everyone's sections are
> suboptimal, then the problem is with fixation and/or processing.
>
> Richard
>
> Richard W. Cartun, MS, PhD
> Director, Histology & The Martin M. Berman, MD Immunopathology &
> Morphologic Proteomics Laboratory
> Director, Biospecimen Collection Programs
> Assistant Director, Anatomic Pathology
> Hartford Hospital
> 80 Seymour Street
> Hartford, CT  06102
> (860) 972-1596
> (860) 545-2204 Fax
>
>
>
> -Original Message-
> From: Martin, Erin via Histonet [mailto:histonet@lists.utsouthwestern.edu]
> Sent: Thursday, October 06, 2016 9:31 AM
> To: histonet
> Subject: [Histonet] Vacuolated and torn?
>
> Good morning everyone!
>
> One of my pathologists says that we are having a problem with the tissue
> on the slides looking vacuolated and torn.  He is convinced it is from
> microtomy.  Anyone have any ideas?  I was thinking that it might be a
> processing issue.
>
>
>
> Thanks in advance!
>
> Erin Martin, Histology Supervisor
> UCSF  Dermatopathology and Oral Pathology Service
> 1701 Divisadero St, San Francisco, CA 94044
> 415-353-7248
>
> Confidentiality Notice
> The information transmitted is intended only for the person or entity to
> which it is addressed and may contain confidential and/or privileged
> material.  Any review, retransmission, dissemination or other use of, or
> taking of any action in reliance upon, this information by persons or
> entities other than the intended recipient is prohibited.  If you receive
> this in error please contact the sender and delete the material from any
> computer.
> ___
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>
> This e-mail message, including any attachments, is for the sole use of the
> intended recipient(s) and may contain confidential and privileged
> information. Any unauthorized review, use, disclosure, or distribution is
> prohibited. If you are not the intended recipient, or an employee or agent
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Re: [Histonet] buffered zn-formalin recipe needed

[Caroline Miller via Histonet]

Histonet archives FTW!

Also IHC world is your friend:
http://www.ihcworld.com/_protocols/histology/zinc_fixative.htm

(and whilst i am here I shall also recommend
http://stainsfile.info/StainsFile/jindex.html as a great friend too)!

mills

On Wed, Sep 28, 2016 at 10:41 AM, Amos Brooks via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi Johanna,
>  The Histonet Archives are your friend. In 2009, Gayle Callis posted a
> great formulation that being the neo-Luddite that I am I printed and have a
> copy of here for just such an occasion...
>
> http://lists.utsouthwestern.edu/mailman/htdig/histonet/
> 2009-October/046985.html
>
> Zinc Fixative (JB Fixative or ZSF)
>
> 0.1M Tris Buffer, pH 7.4
>   Tris Base  12.1 g (TRIZMA)
>   1N HCL --- 81.5 ml
>   Distilled water -- 900 ml
>
>   Mix to dissolve. Adjust pH to 7.4
> Zinc Fixative
>   Calcium Acetate -- 0.5 g
>   Zinc Acetate -- 5.0 g
>   Zinc Chloride -- 5.0 g
>   0.1M Tris Buffer made above -- 1000 ml
> Mix to dissolve. The final pH will be approximately 6.5-7.0. Do not
> readjust
> the pH, as this will cause the zinc to come out of solution. Store Zinc
> Fixative at room temperature. Fix tissues for 24 to 48 hours. Fixation
> longer than 48 hours may make the tissue brittle and difficult to cut.
>
> Good Luck,
>
> Amos
>
>
>
>
> On Wed, Sep 28, 2016 at 12:33 PM,  utsouthwestern.edu
> > wrote:
>
> > Message: 6
> > Date: Tue, 27 Sep 2016 18:21:12 +
> > From: "Preiszner, Johanna" 
> > To: "histonet@lists.utsouthwestern.edu"
> > 
> > Subject: [Histonet] buffered zn-formalin recipe needed
> > Message-ID:
> >  > namprd07.prod.outlook.com>
> >
> > Content-Type: text/plain; charset="iso-8859-1"
> >
> > Hi,
> >
> >
> > For the life of me I can't locate a recipe for buffered zn-formalin, nor
> > online or off.
> >
> >
> > Could someone please help us out?
> >
> >
> > Thanks,
> >
> > Hanna Preiszner
> >
> > ETSU/QCOM
> >
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415 2187297
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Re: [Histonet] GMS

[Caroline Miller via Histonet]

Maybe a fixation issue? Is this happening on all your slides, or just one
set.

Also, do you have a little acetic acid in the light green, I find that
helps. I also find it washes out quickly and a fast dehydration is
necessary.

Very funny that you are having issues with the LG and not the silver, lol,
histology can be so 'interesting' sometimes!

mills

On Mon, Sep 26, 2016 at 4:30 PM, Martin, Gary via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> I am having difficulties with a stain I've done many times ...GMS. The
> light green is only staining the outer edges of the specimen. Any Ideas?
>
>
> Thanks
>
> Gary
>
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Re: [Histonet] Unprocessing tissue

[Caroline Miller via Histonet]

Not crazy at all, this is the kind of thing I was thinking someone would
have out there :)

I have used downey on hard tissue block faces before now too!

thanks so much for all your advice histonet :)

mills

On Fri, Sep 23, 2016 at 12:44 PM, Armstrong, Karoleigh T via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi Caroline,
>
> This may sound a little crazy,
>
> but I was working on some mummified tissue back in the 80's, (Mommy is
> old), and to deal with the dried out tissue we would give them,
>
> 1. A quick .5 percent Sodium Hydroxide soak, 5minutes
>
> 2. Wash is running water, 1 minute
>
> 3. Soak in a 3 parts water 1 part Downey, yes Downey. 5 minutes
>
> 4. Step up to a 1/2 water, 1/2 Downey soak for 5- 10 minutes.
>
> 5. Wash with running water 2-3 minutes.
>
> Proscess your tissue as you usually would.
>
> They came out pretty darn good.
>
> KK Armstrong H.T. (ASCP)
>
>
> --
> Disclaimer: This electronic message may contain information that is
> Proprietary, Confidential, or legally privileged or protected. It is
> intended only for the use of the individual(s) and entity named in the
> message. If you are not an intended recipient of this message, please
> notify the sender immediately and delete the material from your computer.
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Re: [Histonet] Mousse tissue processing

[Caroline Miller via Histonet]

Hi Blanca, This is what I use, note, no formalin on my machine, and you
could probably cut out one of the last alcohols

Step Routine tissue
70% alcohol 10
80% alcohol 10
90% alcohol 20
100% alcohol 20
100% alcohol 20
100% alcohol 30
100% alcohol 30
100% alcohol 30
Xylene 30
Xylene 35
paraffin 30
paraffin 30
paraffin 30
paraffin 30
paraffin at 60oC, P/V on, agit on

On Wed, Sep 21, 2016 at 12:59 PM, Blanca Lopez via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello! Histonets.
> Does anyone has a good protocol for processing mouse tissue? Somebody can
> share.
> thanks
>
> Blanca Lopez
> Histotech (ASCP)
> UTSW Tissue Resource K1.210
> Simmons Comprehensive Cancer Center
> UT Southwestern Medical Center
> Telephone: 214-648-7598
> Email: blanca.lo...@utsouthwestern.edu
>
>
> 
>
> UT Southwestern
>
>
> Medical Center
>
>
>
> The future of medicine, today.
>
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[Histonet] Unprocessing tissue from paraffin

[Caroline Miller via Histonet]

Hi there Histonetters!

I am currently working through some issues of unprocessing tissue to then
restain 'whole mount' with various stains, and then reprocess back to
paraffin.

I am running into issues of tissue being brittle and scratchy tissue after
reprocessing and sectioning.

Can anyone offer any advice on kinder dewaxing procedures and / or
additives to the unprocessing / processing fluids that might help in the
resulting piece of tissue?

I have currently been putting it through the cleaning cycle on the
processor, so standard xylene then alcohol treatment.

thanks in advance for your advice!
mills




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Re: [Histonet] Old scope for donation?

[Caroline Miller via Histonet]

I am sure there are good options on this list, but I might also suggest
AmScope. Not the most perfect of microscopes, but certainly reasonable and
good enough for basic microscopy for kids. Make sure to chose an infinity
corrected optic (i.e. go for a $200 one instead of the $30).

They have staining kits too.

Also the MSA website has a great resource on kids microscopy books:
http://www.microscopy.org/education/projectmicro/searchBooks.cfm

They have a booth as M, I really enjoyed looking at them all. I took
photos of the ones I was interested in purchasing, I am happy to share
these with you also. Let me know

yours,
mills



On Tue, Sep 13, 2016 at 10:55 AM, John Comensky via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Histonetters-
>
> I'm looking for a basic microscope for use with some homeschooling
> children for science and chemistry class. If anybody knows of such a deal
> please email me. Many thanks.
>
> John Comensky, HT(ASCP)QIHC
> jcome...@yahoo.com
>
> Sent from my iPhone
>
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Re: [Histonet] ATM IHC

[Caroline Miller via Histonet]

I have done this in the research world, but I don't have any experience
with clinical analysis of this protein.

I will tell you that cell signalling have the best antibodies in this
space, IMHO:
https://www.cellsignal.com/browse/?Ntk=Products=4294956287=atm+

A company that you can actually follow their protocols and it works! (no
commercial interest)

yours,
mills

On Tue, Aug 16, 2016 at 10:48 AM, Cartun, Richard via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Anyone doing IHC testing for "Ataxia-telangiectasia-mutated protein"
> expression on FFPE tissue?  Thank you.
>
> Richard
>
> Richard W. Cartun, MS, PhD
> Director, Histology & The Martin M. Berman, MD Immunopathology &
> Morphologic Proteomics Laboratory
> Director, Biospecimen Collection Programs
> Assistant Director, Anatomic Pathology
> Hartford Hospital
> 80 Seymour Street
> Hartford, CT  06102
> (860) 972-1596
> (860) 545-2204 Fax
>
>
> This e-mail message, including any attachments, is for the sole use of the
> intended recipient(s) and may contain confidential and privileged
> information. Any unauthorized review, use, disclosure, or distribution is
> prohibited. If you are not the intended recipient, or an employee or agent
> responsible for delivering the message to the intended recipient, please
> contact the sender by reply e-mail and destroy all copies of the original
> message, including any attachments.
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Re: [Histonet] Distilled Water vs DI water for IHC and H staining

[Caroline Miller via Histonet]

Totally agree with the other comments, I had one anecdote though. I worked
in a clinical lab at the Hammersmith in London, with an autostainer. One
day the H started coming off really funny, way too blue, not enough
eosin. We traced everything in the lab, and NOTHING had changed. We
scratched our heads, banged them against the wall and fixed the problem by
changing staining times and fiddling with the protocol.

JUST as we got it fixed the powers that be told us the whole hospital had
changed our main water supply!

Oh, histology, you are so voodoo sometimes!

Happy Friday!

mills

On Thu, Jul 28, 2016 at 9:54 AM, Morken, Timothy via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Charles, for most histology applications water from laboratory-grade DI
> and distilled systems are fairly interchangeable because modern systems
> bring them pretty close to the same purity. But some stains can be affected
> if they are susceptible to chemical interactions with something left in the
> water. That is probably more important for tests like enzyme histochemistry
> than bulk chemical dye staining.
>
> These systems are not just stand-alone "deionized" or "distilled" anymore
> and now include various pre- and post- filtration steps for removing
> particulates, volatile compounds, bacteria, etc  (ours "DI" system has
> several DI resin beds, carbon filters, particulate filters, UV etc). Even
> modern distilling systems will filter tap water with various filters
> (particulates, carbon, bacteria) before distilling.
>
> I don't think the consumer type filters will do the same job as a DI or
> distilled system. They are designed to filter out only the particulates and
> volatile compounds (particulate/bacteria and carbon filters for the most
> part). There is no deionization or demineralization (assuming you are using
> city tap water). However, a reverse osmosis system (which also has several
> other filters pre-RO) may do the trick for you. It is not quite as good at
> purifying as a laboratory-quality DI or Distilled system, but may suffice
> for histology.
>
>
> Tim Morken
> Pathology Site Manager, Parnassus
> Supervisor, Electron Microscopy/Neuromuscular Special Studies
> Department of Pathology
> UC San Francisco Medical Center
>
>
>
>
> -Original Message-
> From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu
> ]
> Sent: Thursday, July 28, 2016 4:54 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Distilled Water vs DI water for IHC and H staining
>
> Does anyone know if there is a difference in staining results when using
> DI water versus Distilled water? Our lab is trying to get away from the DI
> system we have as it is becoming expensive to maintain and we were looking
> to see if we could just use a home drinking filter with UV lighting for our
> water supply. Please let me know any thoughts or concerns you might have
> with this idea
>
> --
>
> Charles Riley HT(ASCP)CM
>
> Histopathology Coordinator/ Mohs
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Re: [Histonet] CD3 antibody for FFPE mouse tissue

[Caroline Miller via Histonet]

+1 to the Dako antibody, it is the only one I have ever found to work (but
I have never used them on the automated staining systems, only old-skool by
hand or sequenza)

mills

On Tue, Jun 21, 2016 at 8:19 AM, Elizabeth Chlipala via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Brett
>
> Dako's rabbit anti human CD3 cross reacts with mouse, or where you looking
> for a CD3 that only detects mouse?
>
> Liz
>
> Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> Premier Laboratory, LLC
> PO Box 18592
> Boulder, CO 80308
> (303) 682-3949 office
> (303) 881-0763 cell
> (303) 682-9060 fax
> l...@premierlab.com
>
> Ship to address:
>
> Premier Laboratory, LLC
> 1567 Skyway Drive, Unit E
> Longmont, CO 80504
> 
> From: Connolly, Brett M via Histonet [histonet@lists.utsouthwestern.edu]
> Sent: Tuesday, June 21, 2016 7:59 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] CD3 antibody for FFPE mouse tissue
>
> Hi all,
>
> Looking for recommendation for an anti-mouse CD3 antibody for FFPE
> sections.
>
> I have some old Histonet messages referring to a Neomarkers RB-360 ab
> which I found through Thermo, but I would like to hear about more recent
> experiences/recommendations
>
> Thanks as always,
> Brett
>
> Brett M. Connolly, Ph.D.
> Prin. Scientist,
> Translational Biomarkers - Imaging
> 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
> information of Merck & Co., Inc. (2000 Galloping Hill Road, Kenilworth,
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> for affiliates is available at
> http://www.merck.com/contact/contacts.html) that may be confidential,
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Re: [Histonet] Static electricity Tip

[Caroline Miller via Histonet]

There is also this thing, that works pretty well, made for record players:

http://www.needledoctor.com/Milty-Zerostat-Gun

mills

On Wed, Jun 1, 2016 at 8:53 AM, Logan, Shannon via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Glad to see this postI also use the dryer balls too (two tennis balls)
> and it helps greatly!! No more static and cutting
> is much more "tame".
> Not to mention that it is healthier because dryer sheets are full of
> chemicals and toxins.
> Have a good one...
>
>
> Shannon H. Logan B.S.  HTL (ASCP)
> Pathology Dept.
> Bellin Health
> 744 S Webster Ave
> Green Bay, WI 54305
> 920-433-3653
>
>
>
> -Original Message-
> From: Cassie P. Davis via Histonet [mailto:
> histonet@lists.utsouthwestern.edu]
> Sent: Wednesday, June 01, 2016 6:29 AM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Static electricity Tip
>
> I cannot be the only histology tech that had a problem with static
> electricity so I am sharing my recent find.
>
> In the past I had tried extra fabric softener, dryer sheets at microtomy,
> wool balls in my dryer with my uniforms, any suggestions, I tried with
> little to no success. The recent try was a large ball of aluminum foil
> (size of a baseball) in the dryer instead for dryer sheets or dryer ball
> with my uniforms (thank you YOUTUBE) and much to my surprise this was the
> first time I was able to go through work without my lab coat or microtomy
> sections sticking to me. Yes, you can reuse it and yes, it does make a
> thudding sound in the dryer that might drive some crazy but no where near
> as crazy as the static at microtomy.
>
>
> Cassandra Davis
> Histology Technician
> Anatomical Pathology Laboratory
> Saint Francis Healthcare
> 701 N. Clayton Street
> Wilmington,DE 19805
> Office:  302-575-8095
> Email:  cda...@che-east.org
> www.saintfrancishealthcare.org
>
> Confidentiality Notice:
> This e-mail, including any attachments is the property of Trinity Health
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Re: [Histonet] No More Blog Posts -- Over and Out!

[Caroline Miller via Histonet]

Thank you Lester, I really appreciate your decision, and I look forward to
reading your histology-related histonet posts.

yours,
mills

On Mon, May 2, 2016 at 10:39 AM, Lester Raff MD via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> To My Lab Colleagues:
>
> As my intent has never been to sow discontent or rancor, I think it is for
> the best if I no longer post links to my blog, lab related or otherwise.
> Of course the blogs go on, and if anyone is interested in being added to my
> mailing list for future notifications, just drop me a line at
> les.r...@post.com   The mailing list is never
> used for any purpose other than announcing a new blog post. Be sure to let
> me know you are from the Histonet list!
>
> I will continue to participate in any histology/pathologist discussions
> here as I have for many years.
>
> Cheers,
>
> Lester J. Raff, MD MBA
> UroPartners
> Medical Director Of Laboratory
> 2225 Enterprise Dr. Suite 2511
> Westchester, Il 60154
> Tel: 708-486-0076
> Fax: 708-492-0203
>
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[Histonet] Paper request please

[Caroline Miller via Histonet]

I can't find this anywhere:
Anal Quant Cytol Histol.

2001
Aug;23(4):291-9.
Quantification of histochemical staining by color deconvolution.
Ruifrok AC

1, Johnston DA

.

Does anyone have a copy please?

Thank you!

mills

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Re: [Histonet] Blog Post Not lab related

[Caroline Miller via Histonet]

Lester, I do believe this is the second 'non lab related' blog post this
week. As we have spoken about before I do not think histonet is an
appropriate place for your blog posts. I, personally, was totally OK with
you co-posting with other lab related topics. However you are now pushing
it and I respectfully ask that you refrain from sending out your blog posts
to the whole list, let people sign up if they are interested.

The issue is that this is a histology related list, if everyone posted
non-lab stuff here it would soon be chaos!

Respectfully yours,
mills

On Wed, Apr 13, 2016 at 11:38 AM, Lester Raff MD via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Just some philosophy towards the end of a long day.
>
>
> http://www.chicagonow.com/downsize-maybe/2016/04/kitten-power-can-get-things-done/
>
>
>
> Just a reminder-I try to limit my blog invitations here. If you enjoy the
> blog, remember to subscribe (no charge, no spam) on the ChicagoNow blog
> site.
>
> Thanks for your readership.
>
> Lester J. Raff, MD MBA
> UroPartners
> Medical Director Of Laboratory
> 2225 Enterprise Dr. Suite 2511
> Westchester, Il 60154
> Tel: 708-486-0076
> Fax: 708-492-0203
>
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>



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[Histonet] Looking for an interesting conversation regarding RNA and Formalin fixation

[Caroline Miller via Histonet]

Hi there Histonet,

I am looking to have a conversation regarding regular histology processing,
mainly formalin fixation, on the RNA species of cell.

I know there is a lot of problem in this space and would like to talk to
someone who is embedded in this world and could offer advice / services to
accurately analyse RNA in formalin fixed, paraffin embedded (and even maybe
resin) tissues.

Asking a lot - yes, I know! However there are a lot of bright folks out
there that might just have an answer for me!

Please PM me!

yours,
mills

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Re: [Histonet] DI water

[Caroline Miller via Histonet]

We use this one:
http://smile.amazon.com/Pure-Stainless-Counter-Distiller-Secure/dp/B003ZJRAWO/ref=pd_sim_sbs_60_12?ie=UTF8=1A4WVXEGE3W42HHT72B0

It has worked so far for our histology applications and our microscopes
waterbath, but I would use 'good' purchased water for any molecular biology
applications. Sigma has some.

You can also get a good deal on 5gallon jugs from mcmastercarr (
http://www.mcmaster.com/#distilled-water/=11fsnq8) of both distilled and
deionized water (about $25 each) - but they cost a lot of money (and
pollution) to deliver. That being said I do not know the carbon footprint
of my distillation machine.

Oh, how I miss my millipore system, but OH MY they cost a lot of money to
buy, install and fix when they go wrong. Too much for this little start up.
Our usage does not afford that luxury!

yours,
mills

On Mon, Mar 7, 2016 at 9:50 AM, Charles Riley via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Does anyone know of a cheap way to make your own DI water? Or a good
> reliable company in the Delaware area that provides DI water services?
>
> --
>
> Charles Riley HT(ASCP)CM
>
> Histopathology Coordinator/ Mohs
>
> Doctors Pathology Services, Dover DE
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Re: [Histonet] cryosectioning undecalcified samples

[Caroline Miller via Histonet]

In this space there is the cryojane (expensive,
http://www.leicabiosystems.com/research/neuroscience/details/product/tape-transfer-system/)
or the Japanese Kawamoto method:
http://section-lab.jp/English/Introduction.htm

I have also found that if you take a regular piece of scotch tape and put
it on the block prior to sectioning that it also works :) (trim to your
area of interest, move the blade along, place on the tape and press down
(use a tool to do this - don't use your hand to reduce the warming of the
tissue, which would give too big a slice), then flip the scotch tape to the
outside of the blade and take a section. You can then stain the tissue on
the tape, dehydrate and clear, then stick the whole thing under a
coverslip. Crude but effective for standard stains. I could not get
immunostaining to work with it though..

It is hard to then get the tissue off the tape - apparently it works with
hexane for the Kawamoto method, but I could never get that to work

Ultimately if you were going to buy something I would say the Kawamoto is a
great place to start, they (he) has a bunch of good tools in cute boxes
that you can work with to make the process easier

Good Luck!

mills



On Wed, Mar 2, 2016 at 11:36 AM, M.O. via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hello,
>
> I am looking into options for cryosectioning undecalcified mouse knees.
> One of my co-workers said he used a film in Japan that stuck to the
> cryoblock and then would do IHC directly on this tape.
>
> Has anyone cut undecalcified cryosections?  If so, what options are there
> and do you recommend one over the other?
>
> Thank you,
> Merissa
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Re: [Histonet] paraformaldehyde

[Caroline Miller via Histonet]

I don't know if there is a hard and fast rule, but I keep 20% in the fridge
for around 6 months or until the little bits start to form on the bottom.
4% I would never keep that long, maybe a week at most.

Best if you want fresh, small volumes, all the time is to get those little
37% ampules and dilute as necessary

I am very interested to hear other folks opinions too, as I am sure they
will vary!

yours,
mills

On Thu, Feb 25, 2016 at 6:11 AM, Bruijntjes, J.P. (Joost) via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> Hi all
>
> Can anyone tell me what the expiration time is for home-made
> paraformaldehyde?
>
> Joost
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Re: [Histonet] Shrinkage

[Caroline Miller via Histonet]

hi All, I have done some experiments in this area for mouse brains, and I
find that there is actually an expansion of tissue after formalin fixation
(around 10%), but then certainly a shrinkage to 100% dehydration agent of
about 20% from the original size. We found similar results with alcohol,
acetone and THF.

This is manual fluid changes or a day per solution, with no vacuum, temp or
pressure.

Happy to share the data if anyone is interested

yours,
mills

On Mon, Feb 22, 2016 at 1:10 PM, Terri Braud via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> LOL...Shrinkage...heh, heh.
> But seriously, there should be little to no gross shrinkage from formalin
> fixation and if the specimen is properly fixed, then there should be very
> little gross shrinkage as it is dehydrated.  That is supposed to be the
> point!  If someone is getting 30% shrinkage, there is something seriously
> wrong with their processing schedule.
> Sincerely, Terri
> Terri L. Braud, HT(ASCP)
> Anatomic Pathology Supervisor
>
> Today's Topics:
>2. formalin and shrinkage (Gudrun Lang)
>
>
> Message: 2
> Date: Mon, 22 Feb 2016 16:59:21 +0100
> From: "Gudrun Lang" 
> Subject: [Histonet] formalin and shrinkage
> Hi!
> Today someone asked me about shrinkage caused by the fixation with
> formaldehyde specially on skin-biopsies.  She spoke about shrinkage of 30%
> percent. In my opinion shrinkage is mainly caused by the processing with
> dehydration and defatting. Formaldehyde renders the tissue harder but not
> strictly smaller.
> What is the opinion of the community?
> Gudrun
>
>
>
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Re: [Histonet] Double stain IHC question

[Caroline Miller via Histonet]

+ to Gudrun's comments.

My addition is that fluorescence labels may work for this application.
Again, depending on the species of the antibodies. The other advantage of
the fluorescence is that you would be able to truly see the colocalization
(yellow for instance if you used red and green fluorescent antibodies).
Rather than the muddiness I have often got when trying two antibodies in
bright field chromagenic stains.

Remember though autofluorescence is greatly increased with paraffin
tissues, that may put another issue on your plate.

Good luck!

yours,
mills

On Sat, Feb 20, 2016 at 3:47 AM, Gudrun Lang via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> In my opinion, this would only be possible, if the commercial and the
> homegrown antibody are from different species. For example one from mouse
> and one from rabbit.
> Then you can proceed with different secondaries (goat anti mouse conjugated
> with peroxidase, goat anti rabbit conjugated with alkaline phosphatase).
> Then chromogens that work with each of the enzymes.
>
> If the antibodies are from the same species I see no way to distinguish
> both. Only if one is conjugated with biotin and the other with digoxigenin,
> then you could proceed with secondaries against biotin and digoxigenin.
> etc..
>
> Gudrun
>
> -Ursprüngliche Nachricht-
> Von: Judi Ford via Histonet [mailto:histonet@lists.utsouthwestern.edu]
> Gesendet: Samstag, 20. Februar 2016 01:54
> An: histonet@lists.utsouthwestern.edu
> Betreff: [Histonet] Double stain IHC question
>
> Hi everyone,
> I have a question in chromogenic double staining. Here is the situation.
> Tissue = human, frozen
> Antibody = same protein (A)
>
> 1.   Commercial antibody of A
>
> 2.   Homegrown antibody of A, human, biotinylated
>
> Question: can you stain both versions of this antibody on the same tissue,
> same slide? Goal is to see where each stains in the tissue and if they
> co-localize. If they do co-localize then how do you distinguish between
> that
> and where they stain individually? Would you use different chromogens and
> hope that where they come together it turns a different color?
> I am really interested if this can work. Thanks in advance for any replies.
> Judi
> South San Francisco, CA
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Re: [Histonet] Tissue processing question

[Caroline Miller via Histonet]

I really like this type:
https://www.fishersci.com/shop/products/starplex-scientific-histoplex-tissue-cassettes-micromesh-chamber-8/p-2782584

(although I buy them from mastertech, but they seem to have dissapeared
from their website)

They are great for both large tissues, and also biopsies. A long time ago
when I worked in a clinical lab we used the tissue paper and I found that
if everything was not heated just right the biopsies would stick and things
like currettes were hard to scrape up from there, I always thought I was
doing the tissue damage

yours
mills

On Fri, Jan 29, 2016 at 10:06 AM, Walter Benton via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

> We use hair wrapping paper used for perms. It is the same paper called
> "biopsy wraps," but at a significant price reduction. You can buy a variety
> of sizes and the wraps do not cause artifacts and are porous enough for
> ample solution penetration. Biopsy paper comes in blue and other colors,
> but the hair wraps only come in white. Our overall experience with them has
> been great.
>
> Let me know if you need any other information.
>
>
> Walter Benton HT(ASCP)QIHC
> Lab Operations Manager
> Chesapeake Urology Associates
> 806 Landmark Drive, Suite 127
> Glen Burnie, MD 21061
> 443-471-5850 (Direct)
> 410-768-5961 (Lab)
> 410-768-5965 (Fax)
> Chesapeakeurology.com
>
> Voted a Best Place to Work by
> Baltimore and Modern Healthcare
> Magazines.
>
>
>
> -Original Message-
> From: Charles Riley via Histonet [mailto:histonet@lists.utsouthwestern.edu
> ]
> Sent: Friday, January 29, 2016 12:43 PM
> To: histonet@lists.utsouthwestern.edu
> Subject: [Histonet] Tissue processing question
>
> Hello all,
>
>  I was wondering what everyone uses to secure biopsy and scant tissues
> through processing. Also what would you recommend placing breast cores in
> for processing. Having an argument with grossing staff and pathologist
> about whether to use sponges, tissue paper, or something else. Looking for
> the best option that will allow for reagents to penetrate tissue and not
> leave any artifact
>
> --
>
> Charles Riley HT(ASCP)CM
>
> Histopathology Coordinator/ Mohs
>
> Doctors Pathology Services, Dover DE
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> intended recipient, you are hereby notified that any dissemination,
> distribution or copying of this transmission is strictly prohibited. If you
> have received this transmission in error, please notify the transmitting
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Director of Histology
3Scan.com
415 2187297
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Re: [Histonet] Thanks...also blog post

[Caroline Miller via Histonet]

Thank you Lester, this is a great middle ground! As someone who was a
little put out by your totally off-topic emails, I am totally OK with you
promoting your blog post inside a legitimate histonet post.

Good luck with your issues.

yours,
mills

On Thu, Jan 21, 2016 at 12:03 PM, Lester Raff MD via Histonet <
histonet@lists.utsouthwestern.edu> wrote:

>
> http://www.chicagonow.com/downsize-maybe/2016/01/can-someone-create-this-app/
>
>
> Thanks to the suggestions for flooring. We are finding the concept of
> moving all our block and slide cabinets to allow us to lay new flooring
> overwhelming. We may opt to have the housekeeping crew put on a new coating
> that supposedly is more slip resistant. WE are also still researching our
> disposal of 10 year old blocks. Thanks for the suggestions.
>
>
> Lester J. Raff, MD MBA
> UroPartners
> Medical Director Of Laboratory
> 2225 Enterprise Dr. Suite 2511
> Westchester, Il 60154
> Tel: 708-486-0076
> Fax: 708-492-0203
>
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415 2187297
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[Histonet] Paper request

[Caroline Miller via Histonet]

Hi All, Does anyone have access to this paper:
Toluidine Blue-Alizarin Red S Staining of Cartilage and Bone in Whole-Mount
Skeletons in Vitro


Stain Technology Volume 40
, Issue 2
, 1965


Thanks in advance, I hope you are all doing well!


yours,

mills

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415 2187297
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Re: [Histonet] Paper request

[Caroline Miller via Histonet]

Got it! Thanks Histonet!! :)

mills

On Thu, Jan 14, 2016 at 2:59 PM, Caroline Miller  wrote:

> Hi All, Does anyone have access to this paper:
> Toluidine Blue-Alizarin Red S Staining of Cartilage and Bone in
> Whole-Mount Skeletons in Vitro
>
>
> Stain Technology Volume 40
> , Issue 2
> , 1965
>
>
> Thanks in advance, I hope you are all doing well!
>
>
> yours,
>
> mills
>
> --
> Caroline Miller (mills)
> Director of Histology
> 3Scan.com
> 415 2187297
>



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Director of Histology
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415 2187297
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Re: [Histonet] Off topic posts

[Caroline Miller via Histonet]

+1 to Rene and Anne, these posts have nothing to do with histology and I wish 
they would go away.

Dr Raff, please go post them somewhere more relevant.

Yours,
mills



Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

> On Dec 21, 2015, at 6:18 AM, Rene J Buesa via Histonet 
>  wrote:
> 
> So do I!
> René 
> 
>On Monday, December 21, 2015 12:28 AM, Anne Van Binsbergen via Histonet 
>  wrote:
> 
> 
> Dear Lester Raff MD
> Yes some of us do visit the Histonet over weekends. 
> Your posts are so annoying and self-serving. 
> Please go away!
> Thanks
> Annieinarabia 
> 
> Sent from my iPhone
> 
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> 
> 
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Re: [Histonet] : Are gloves required when cutting FFPE blocks?

[Caroline Miller via Histonet]

I tend to leave my squames  on the slide, so I always wear gloves too

mills


Caroline Miller (mills)
Director of Histology
3Scan, Inc
415-2187297

> On Nov 27, 2015, at 5:50 AM, White, Lisa M. via Histonet 
>  wrote:
> 
> I am not aware of any regulation to use gloves to section.
> 
> 
> 
> However I do use them to section on the microtome, I got really tired of
> picking human out from under my fingernails.  
> 
> The other 4 HT that I work with do not use them on standard microtome
> sections.
> 
> 
> 
> CJD would be the only exception, but then again the all of the
> processing equipment would then be a problem also.
> 
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[Histonet] Tissue processor advice for a small start-up

[Caroline Miller via Histonet]

Hi All,

Our company has reached the size where we can justify buying our own
processor! Yippee!!

However that also leads me to the issue of which one to buy :)

We will have very small batches of blocks to process, approx 10 per process
maybe twice per week, but we need the machine to be very responsive to
different processes - we have a microscope that sections and images at the
same time, therefore we do the staining prior to processing, and have to
tweak the protocols to keep enough contrast int the tissue by the end.

In short:
- low volume
- easy to use and change protocols
- good support for servicing and fixing should things go wrong.
- No formalin will touch the machine, and we will want to change out
solvents from the standard alcohols and xylenes (therefore I need a machine
who's warranty would not be affected by this.
- I would love to get this all in one package and buy the service contract
at the same time as the machine, preferably with the same company.
- If it helps with the cost, then I also want to buy a paraffin microtome :)

We are in San Francisco.

Commercial replies are totally acceptable here, but please do not reply all.

Thanks, as ever, histonet!

yours,
mills

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Director of Histology
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415 2187297
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