[Histonet] RE: Eliminating the edge effect in IHC/IF

Tue Jan 12 15:03:13 CST 2010

I do my immuno staining manually using ProbeOn Plus slides which employ the capillary action principle.  I always monitor the taking up/whicking off of my solutions for each pair of slides throughout the entire staining process and know that my sections never dry out (I incubate using moist chambers with plenty of fluid).  I work at a research/diagnostic medical school facility where our primary focus is on experimental tissues, using standardized and rigid guidelines for fixation: 24 hours at 4 degrees overnight in 4% PF, followed by post fixation in 70% ethanol for a few days.  Never see the "edge effect" with these sections.  However, I do occasionally see the effect using archival diagnostic samples that were fixed in 10% NBF for a minimum of 48 hours, and probably longer. I have therefore always perceived this effect to be caused by extended, rather than incomplete fixation.

Hermina

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Tuesday, January 12, 2010 3:41 PM
To: C.M. van der Loos; histonet <@t> lists.utsouthwestern.edu; MargaretSherwood
Cc: kcai <@t> prosci-inc.com
Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF

Again, provided that you are doing IHC manually.
René J.

--- On Tue, 1/12/10, Sherwood, Margaret <MSHERWOOD <@t> PARTNERS.ORG> wrote:

From: Sherwood, Margaret <MSHERWOOD <@t> PARTNERS.ORG>
Subject: RE: [Histonet] RE: Eliminating the edge effect in IHC/IF
To: "C.M. van der Loos" <c.m.vanderloos <@t> amc.uva.nl>, histonet <@t> lists.utsouthwestern.edu
Cc: kcai <@t> prosci-inc.com
Date: Tuesday, January 12, 2010, 3:28 PM

Actually, when I read about the problem, my thought was that if you don't circle
your tissue with an immunopen or wax pencil, then the reagents (esp. primary
antibody)might not cover the tissue evenly as you say.  So I tend to agree with
you.

Peggy 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of C.M. van der
Loos
Sent: Tuesday, January 12, 2010 2:55 PM
To: histonet <@t> lists.utsouthwestern.edu
Cc: kcai <@t> prosci-inc.com
Subject: [Histonet] RE: Eliminating the edge effect in IHC/IF

Hi all,We also have observed this phenomenon many times. But sorry Colleen and
Rene, I don't believe that an fixation issue is the explanation why the edges
are sometimes stronger than the rest. To my opinion this is a bit too easy. One
of my explanations is that the immuno reagents tend to stick to the edges of the
tissue section, especially when no Tween20 is involved. As a result the outer
edges might become a little dry during incubation times and cause darker mostly
non-specific staining. Always try to cover a section completely, including a rim
around the section. However, to be honest, I am sure my explanation is certainly
not always appropriate. Anyone else????Cheers,ChrisChris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
phone:  +31 20 5665631
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet <@t> lists.utsouthwestern.edu, Karen Cai <kcai <@t> prosci-inc.com>

Usually that is the result of incomplete fixation. Check your fixation protocol.
Ren  J.

--- On Mon, 1/11/10, Karen Cai <kcai <@t> prosci-inc.com> wrote:

From: Karen Cai <kcai <@t> prosci-inc.com>
Subject: [Histonet] Eliminating the edge effect in IHC/IF
To: histonet <@t> lists.utsouthwestern.edu
Date: Monday, January 11, 2010, 2:00 PM

Hi,
I have a question for the generous input. When I do the IHC or IF, it
seems very common that the intensity of the edge area of the tissue is
always stronger than the central tissue part. Is it possible to
eliminate this and make the staining evenly distributed around the whole
tissue section?

Your kind help is greatly appreciated,

Thanks in advance,

Best,
Karen

Karen Cai
Research Scientist
Prosci Incorporated
(858) 513-2638 x 204
(858) 513-2692 Fax
<http://www.prosci-inc.com> www.prosci-inc.com
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