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

[Karen Cai]

Hello,
Thank you very much for the help. I tried some methods and found that fixation plays major role in my case. 4% PFA followed with ETOH fixation improves.
 
Just want to share my experience here.
 
 
 
Best,
Karen
 
 
 
-----Original Message-----
From: Rene J Buesa [mailto:rjbuesa <@t> yahoo.com] 
Sent: Tuesday, January 12, 2010 12:40 PM
To: histonet <@t> lists.utsouthwestern.edu; C.M. van der Loos
Cc: kcai <@t> prosci-inc.com; cforster <@t> umn.edu
Subject: RE: Eliminating the edge effect in IHC/IF
 

Hi Dr. van der Loos:
I had also experienced that artifact caused by less than necessary reagents, but that happens mostly when IHC is done manually, or when the autostainer delivers less amount than required or programmed.
IF we assume that the colleague with the question did the IHC manually, your explanation can be accepted, otherwise, poor fixation is a valid cause to the problem.
René J.

--- On Tue, 1/12/10, C.M. van der Loos <c.m.vanderloos <@t> amc.uva.nl> wrote:

From: C.M. van der Loos <c.m.vanderloos <@t> amc.uva.nl>
Subject: RE: Eliminating the edge effect in IHC/IF
To: histonet <@t> lists.utsouthwestern.edu
Cc: kcai <@t> prosci-inc.com, rjbuesa <@t> yahoo.com, cforster <@t> umn.edu
Date: Tuesday, January 12, 2010, 2:54 PM
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,
Chris
Chris 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 <http://www.prosci-inc.com/> > www.prosci-inc.com