[Histonet] IHC Slides

2012-03-12 Thread Courtney Pierce
Having problems with IHC slides not looking the same between runs. Does it have 
to do with how long and at what temp they are in the oven for?

Courtney Pierce
IHC Specialist
Quintiles
Translational RD - Oncology
Innovation
Navigating the new health

610 Oakmont Lane
Westmont, IL 60559

Office: + 630-203-6234
courtney.pie...@quintiles.com

clinical | commercial | consulting | capital


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Re: [Histonet] IHC Slides

2012-03-12 Thread Rene J Buesa
Not really. Time in the oven and temp is usually not an issue as long as the 
tissue is well fixed/processed/infiltrated.
René J.

--- On Mon, 3/12/12, Courtney Pierce courtney.pie...@quintiles.com wrote:


From: Courtney Pierce courtney.pie...@quintiles.com
Subject: [Histonet] IHC Slides
To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu
Date: Monday, March 12, 2012, 3:35 PM


Having problems with IHC slides not looking the same between runs. Does it have 
to do with how long and at what temp they are in the oven for?

Courtney Pierce
IHC Specialist
Quintiles
Translational RD - Oncology
Innovation
Navigating the new health

610 Oakmont Lane
Westmont, IL 60559

Office: + 630-203-6234
courtney.pie...@quintiles.com

clinical | commercial | consulting | capital


**  IMPORTANT--PLEASE READ  
This electronic message, including its attachments, is COMPANY CONFIDENTIAL
and may contain PROPRIETARY or LEGALLY PRIVILEGED information.  If you are 
not the intended recipient, you are hereby notified that any use, disclosure,
copying, or distribution of this message or any of the information included
in it is unauthorized and strictly prohibited.  If you have received this
message in error, please immediately notify the sender by reply e-mail and
permanently delete this message and its attachments, along with any copies
thereof. Thank you. 


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[Histonet] IHC slides

2011-03-02 Thread sgoebel
So...I have run down slides and done antigen retrieval on my FFPE
slides.  They are currently in antigen retrieval that has come to room
temperature.  I am not going to be able to finish the IHC stains until
tomorrow.  Will it be better to keep the slides in the antigen retrieval
solution at 4 degrees overnight, or put it in buffer at 4 degrees
overnight?  I really don't want to stay here late.

Thanks histo-hotties!!

 

Sarah Goebel, BA, HT(ASCP)

Histotechnologist

Mirna Therapeutics

2150 Woodward Street

Suite 100

Austin, Texas  78744

(512)901-0900 ext. 6912

 

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RE: [Histonet] IHC slides

2011-03-02 Thread Sebree Linda A
We've held slides post-retrieval in buffer (tris, PBS, etc.) overnight
with no problem. 


Linda A. Sebree
University of Wisconsin Hospital  Clinics
IHC/ISH Laboratory
DB1-223 VAH
600 Highland Ave.
Madison, WI 53792
(608)265-6596


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
sgoe...@mirnarx.com
Sent: Wednesday, March 02, 2011 1:24 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC slides

So...I have run down slides and done antigen retrieval on my FFPE
slides.  They are currently in antigen retrieval that has come to room
temperature.  I am not going to be able to finish the IHC stains until
tomorrow.  Will it be better to keep the slides in the antigen retrieval
solution at 4 degrees overnight, or put it in buffer at 4 degrees
overnight?  I really don't want to stay here late.

Thanks histo-hotties!!

 

Sarah Goebel, BA, HT(ASCP)

Histotechnologist

Mirna Therapeutics

2150 Woodward Street

Suite 100

Austin, Texas  78744

(512)901-0900 ext. 6912

 

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[Histonet] IHC slides

2011-03-02 Thread Mehmet Fatih BOZKURT
You can put them %3 H2O2 for 10 minutes. and then drop primary antibody and
incubate at 4 degrees overnight.
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RE: [Histonet] IHC slides

2011-03-02 Thread Mike Pence
Just hold them in buffer overnight.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of
sgoe...@mirnarx.com
Sent: Wednesday, March 02, 2011 1:24 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC slides


So...I have run down slides and done antigen retrieval on my FFPE
slides.  They are currently in antigen retrieval that has come to room
temperature.  I am not going to be able to finish the IHC stains until
tomorrow.  Will it be better to keep the slides in the antigen retrieval
solution at 4 degrees overnight, or put it in buffer at 4 degrees
overnight?  I really don't want to stay here late.

Thanks histo-hotties!!

 

Sarah Goebel, BA, HT(ASCP)

Histotechnologist

Mirna Therapeutics

2150 Woodward Street

Suite 100

Austin, Texas  78744

(512)901-0900 ext. 6912

 

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Re: [Histonet] IHC slides

2011-03-02 Thread Rene J Buesa
In buffer at 4ºC overnight
René J.

--- On Wed, 3/2/11, sgoe...@mirnarx.com sgoe...@mirnarx.com wrote:


From: sgoe...@mirnarx.com sgoe...@mirnarx.com
Subject: [Histonet] IHC slides
To: histonet@lists.utsouthwestern.edu
Date: Wednesday, March 2, 2011, 2:24 PM


So...I have run down slides and done antigen retrieval on my FFPE
slides.  They are currently in antigen retrieval that has come to room
temperature.  I am not going to be able to finish the IHC stains until
tomorrow.  Will it be better to keep the slides in the antigen retrieval
solution at 4 degrees overnight, or put it in buffer at 4 degrees
overnight?  I really don't want to stay here late.

Thanks histo-hotties!!



Sarah Goebel, BA, HT(ASCP)

Histotechnologist

Mirna Therapeutics

2150 Woodward Street

Suite 100

Austin, Texas  78744

(512)901-0900 ext. 6912



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RE: [Histonet] ihc slides

2011-01-24 Thread Settembre, Dana
Hi Cindy,
That too has happened to me and the process that you do to remedy it,
works for me too. (take off coverslip properly, re-apply DAB, and you
get a reaction.) My experience is that the slides are not shot, as described 
by another Histonetter.
There was definitely something else going on.
I work with the Dako too.
Is it possible that the Autostainer was not programmed to apply
antibody in the crucial zones?
Look into that possibility.
Good Luck.

Dana Settembre
University Hospital - UMDNJ
Newark, NJ  

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Cynthia Pyse
Sent: Monday, January 24, 2011 7:58 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] ihc slides

Happy Monday Histonetters

I need to pick the brains of the IHC experts out there in histoland. I run
IHC on the Dako autostainer. On one of our runs there was not enough DAB to
dispense on all the slides. My tech dehydrated the slides and cover slipped
them. We printed the stain log on the run and confirmed that the DAB was not
dispensed on three slides. The cover slips were removed and the slide were
soaked in xylene to remove the cover slipping media. The slides were
rehydrated and DAB was applied. There was still no reaction after 10
minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can
anyone out there give me a reason that the slides would not react after the
reapplication of the DAB. I have done this before when the DAB solution was
short on the run and it worked. I can't figure out why this didn't react. I
called tech service at Dako and talked to the supervisor, she agrees with me
that the reapplication should have worked. I checked the run log and the
only missing step is the DAB. The antibodies with the missing DAB have their
own controls on the slides. Any thought out there as to what the problem is.
I have had the PM done on the Dako which should resolve the problem
according to Dako. There has not been a problem since. This mystery will
drive me crazy until I can figure out why. I could use help. Thanks in
advance, I appreciate any input.

Cindy

 

Cindy Pyse, CLT, HT (ASCP)

Laboratory/Histology Supervisor

X-Cell Laboratories

716-250-9235

e-mail cp...@x-celllab.com

 

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Re: [Histonet] ihc slides

2011-01-24 Thread Greg Dobbin
Cynthia,
Hematoxylin will inactivate the peroxidase (link) in the tissues. You must go 
back and re-do the link step and hopefully the antigenic sites will still 
accept more streptavidin (or similar) conjugated antibody.
Cheer.
Greg
 
Greg Dobbin, R.T.
Chief Technologist, Anatomic Pathology
Dept. of Laboratory Medicine,
Queen Elizabeth Hospital,
P.O. Box 6600
Charlottetown, PEC1A 8T5
Phone: (902) 894-2337
Fax: (902) 894-2385
 
I find that the harder I work, the 
more luck I seem to have.
- Thomas Jefferson


 Cynthia Pyse cp...@x-celllab.com 1/24/2011 8:57 AM 
Happy Monday Histonetters

I need to pick the brains of the IHC experts out there in histoland. I run
IHC on the Dako autostainer. On one of our runs there was not enough DAB to
dispense on all the slides. My tech dehydrated the slides and cover slipped
them. We printed the stain log on the run and confirmed that the DAB was not
dispensed on three slides. The cover slips were removed and the slide were
soaked in xylene to remove the cover slipping media. The slides were
rehydrated and DAB was applied. There was still no reaction after 10
minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can
anyone out there give me a reason that the slides would not react after the
reapplication of the DAB. I have done this before when the DAB solution was
short on the run and it worked. I can't figure out why this didn't react. I
called tech service at Dako and talked to the supervisor, she agrees with me
that the reapplication should have worked. I checked the run log and the
only missing step is the DAB. The antibodies with the missing DAB have their
own controls on the slides. Any thought out there as to what the problem is.
I have had the PM done on the Dako which should resolve the problem
according to Dako. There has not been a problem since. This mystery will
drive me crazy until I can figure out why. I could use help. Thanks in
advance, I appreciate any input.

Cindy



Cindy Pyse, CLT, HT (ASCP)

Laboratory/Histology Supervisor

X-Cell Laboratories

716-250-9235

e-mail cp...@x-celllab.com



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Re: [Histonet] ihc slides

2011-01-24 Thread Greg Dobbin
I should add that the DAB step may also have quenched all of the linked 
peroxidases even before the hematoxilin was added (which would finish off any 
that remained).
Greg
 
Greg Dobbin, R.T.
Chief Technologist, Anatomic Pathology
Dept. of Laboratory Medicine,
Queen Elizabeth Hospital,
P.O. Box 6600
Charlottetown, PEC1A 8T5
Phone: (902) 894-2337
Fax: (902) 894-2385
 
I find that the harder I work, the 
more luck I seem to have.
- Thomas Jefferson


 Cynthia Pyse cp...@x-celllab.com 1/24/2011 8:57 AM 
Happy Monday Histonetters

I need to pick the brains of the IHC experts out there in histoland. I run
IHC on the Dako autostainer. On one of our runs there was not enough DAB to
dispense on all the slides. My tech dehydrated the slides and cover slipped
them. We printed the stain log on the run and confirmed that the DAB was not
dispensed on three slides. The cover slips were removed and the slide were
soaked in xylene to remove the cover slipping media. The slides were
rehydrated and DAB was applied. There was still no reaction after 10
minutes, The DAB was reapplied for up to 30 minutes with no reaction. Can
anyone out there give me a reason that the slides would not react after the
reapplication of the DAB. I have done this before when the DAB solution was
short on the run and it worked. I can't figure out why this didn't react. I
called tech service at Dako and talked to the supervisor, she agrees with me
that the reapplication should have worked. I checked the run log and the
only missing step is the DAB. The antibodies with the missing DAB have their
own controls on the slides. Any thought out there as to what the problem is.
I have had the PM done on the Dako which should resolve the problem
according to Dako. There has not been a problem since. This mystery will
drive me crazy until I can figure out why. I could use help. Thanks in
advance, I appreciate any input.

Cindy



Cindy Pyse, CLT, HT (ASCP)

Laboratory/Histology Supervisor

X-Cell Laboratories

716-250-9235

e-mail cp...@x-celllab.com



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RE: [Histonet] ihc slides

2011-01-24 Thread Margaryan, Naira
Hi Cynthia,

I agree with Greg, you have to re-do your staining from beginning (from AR 
step) after removing the hematoxilin.

Naira 

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[Histonet] IHC Slides, Hotplate vs Oven

2010-04-21 Thread Gagnon, Eric
Tony's answer (62 degrees C for 25 mins) is the minimum to ensure section 
adheres to slide.  We all have to stain slides that have been baked longer than 
30 mins.  However, especially for Her2, longer baking times can result in false 
negatives or at least diminished staining.  The justification for baking longer 
is an attempt to prevent the section washing off or lifting.  We are seeing 
evidence that section adhesion is not a good tradeoff for diminished staining.  
Ideally both can be achieved with no tradeoff.
 
Tony or others who may wish to answer:
 
1. How do you monitor the shortened baking time, especially when multiple racks 
of slides are cut throughout the day?
2. Assuming charged slides and cell conditioning/antigen retrieval are 
employed, are you seeing major problems with section adhesion?
3. What if slides are received from another institution for staining, or slides 
get baked longer than 25 mins?
 
Thanks in advance,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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RE: [Histonet] IHC Slides, Hotplate vs Oven

2010-04-21 Thread Tony Henwood
Eric,

Some of my thoughts on your discussion questions:

1. How do you monitor the shortened baking time, especially when
multiple racks of slides are cut throughout the day?
We have timers set for when each rack goes into the oven. This might be
an issue for labs with larger batches (we are a Children's hospital)

 2. Assuming charged slides and cell conditioning/antigen retrieval are
employed, are you seeing major problems with section adhesion?
No, not usually. Problem sections tend to be those that are inadequately
fixed ( therefore inadequately processed), brain and bone sections.
They may tend to lift.

 3. What if slides are received from another institution for staining,
or slides get baked longer than 25 mins? 
Again, it is difficult to know the conditions that the tissues have
undergone. In-built controls, if present are invaluable.
We use a BondMax immunostainer, so the antigen retrieval temperature and
time are better controlled than is possible with the manual procedure
(or when I do it!).

I believe that incomplete fixation causes more problems for morphology
and immunohistochemistry than is commonly appreciated. 

Fix the fixing and most problems are solved - Hows that for a sweeping
statement!

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager  Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Gagnon,
Eric
Sent: Thursday, 22 April 2010 5:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Slides, Hotplate vs Oven


Tony's answer (62 degrees C for 25 mins) is the minimum to ensure
section adheres to slide.  We all have to stain slides that have been
baked longer than 30 mins.  However, especially for Her2, longer baking
times can result in false negatives or at least diminished staining.
The justification for baking longer is an attempt to prevent the section
washing off or lifting.  We are seeing evidence that section adhesion is
not a good tradeoff for diminished staining.  Ideally both can be
achieved with no tradeoff.
 
Tony or others who may wish to answer:
 
1. How do you monitor the shortened baking time, especially when
multiple racks of slides are cut throughout the day? 2. Assuming charged
slides and cell conditioning/antigen retrieval are employed, are you
seeing major problems with section adhesion? 3. What if slides are
received from another institution for staining, or slides get baked
longer than 25 mins?
 
Thanks in advance,
 
Eric Gagnon MLT
Histology Laboratory
Kingston General Hospital
Kingston, Ontario, Canada
 
 


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[Histonet] IHC Slides, Hotplate vs Oven

2010-04-20 Thread Laurie Colbert
I'm curious as to whether or not others put their IHC slides on a
hotplate after cutting?  If so, at what temperature and for how long?
Who only uses an oven - and what temp and for how long?  Does anyone use
both the hotplate and the oven?

 

Laurie Colbert

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RE: [Histonet] IHC Slides, Hotplate vs Oven

2010-04-20 Thread Mike Pence
We use an oven only at 60c for 20 minutes.

-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie
Colbert
Sent: Tuesday, April 20, 2010 9:40 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Slides, Hotplate vs Oven


I'm curious as to whether or not others put their IHC slides on a
hotplate after cutting?  If so, at what temperature and for how long?
Who only uses an oven - and what temp and for how long?  Does anyone use
both the hotplate and the oven?

 

Laurie Colbert

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RE: [Histonet] IHC Slides, Hotplate vs Oven

2010-04-20 Thread Sebree Linda A
I sometimes put cut slides on a warm plate attached to my water bath.
One of the plates is set at 55d and the other at 60d.  I leave them till
all the paraffin is completely melted.   I only do this for cases I'm
paranoid about staying on! 


Linda A. Sebree
University of Wisconsin Hospital  Clinics
IHC/ISH Laboratory
DB1-223 VAH
600 Highland Ave.
Madison, WI 53792
(608)265-6596


-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie
Colbert
Sent: Tuesday, April 20, 2010 9:40 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Slides, Hotplate vs Oven

I'm curious as to whether or not others put their IHC slides on a
hotplate after cutting?  If so, at what temperature and for how long?
Who only uses an oven - and what temp and for how long?  Does anyone use
both the hotplate and the oven?

 

Laurie Colbert

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RE: [Histonet] IHC Slides, Hotplate vs Oven

2010-04-20 Thread Tony Henwood
We use an oven (62oC) for 25min only. See:

Henwood, A., (2005) Effect of Slide Drying at 80°C on Immunohistochemistry J 
Histotechnol 28(1):45-46.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager  Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-Original Message-
From: histonet-boun...@lists.utsouthwestern.edu 
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Laurie Colbert
Sent: Wednesday, 21 April 2010 12:40 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC Slides, Hotplate vs Oven


I'm curious as to whether or not others put their IHC slides on a hotplate 
after cutting?  If so, at what temperature and for how long? Who only uses an 
oven - and what temp and for how long?  Does anyone use both the hotplate and 
the oven?

 

Laurie Colbert

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This email and any files transmitted with it are confidential and intended 
solely for the use of the individual or entity to whom they are addressed. If 
you are not the intended recipient, please delete it and notify the sender.

Views expressed in this message and any attachments are those of the individual 
sender, and are not necessarily the views of The Children's Hospital at Westmead

This note also confirms that this email message has been virus scanned and 
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accepts no liability for any consequential damage resulting from email 
containing computer viruses.
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