[Histonet] RE: Histonet@lists.utsouthwestern.edu
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Wed Jan 11 12:54:02 CST 2006
Hi Gayle and others,
Just some remarks on the methanol-issue from my perspective:
Basically you are right that methanol is nasty stuff for many CD
markers. Using methanol on human cryostat sections has a deleterious
effect on far most antigens (yes, I am aware that the mouse-world
thinks different on this). However, with FFPE sections that have been
through alcohol-steps several times a methanol step doesn't seem to
matter that much anymore. Once an antigen or epitope has survived
embedding, methanol will not do any harm. All my FFPE
sections developed for peroxidase activity are blocked with methanol +
0.3% peroxide for 20 min at RT (Streefkerk, 1972 in JHC) without any
problem. Some people claim there are exceptions here, but so far I
haven't seen real comparison experiments proving this.
Blocking of endogenous peroxidase activity with methanol+0.3% peroxide
is far more effective than PBS+03.% peroxide+0.1% sodium azide. The
latter just blocks erythrocyte pseudoperoxidase but just "weaken" the
endogenous peroxide activity in neutrophils.
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
Date: Wed, 11 Jan 2006 08:42:11 -0700
From: Gayle Callis <gcallis <@t> montana.edu>
Subject: [Histonet] Methanol and CD markers
To: Lori Richey <lrichey <@t> u.washington.edu>,
Histonet <@t> lists.utsouthwestern.edu
Just a note here. Methanol is known to create problems with CD marker
immunohistochemistry and that is why many use hydrogen peroxide in
buffer
instead of this solvent. This point was brought up in Jules Elias's
book
on immunohistochemistry and also on Histonet in the past. (see
archives).
At 04:02 PM 1/10/2006, you wrote:
>We use both CD4 and CD8 on FFPE sections. We had problems with the
CD4
>until we changed our quenching step to 0.5% H2O2 in MEOH for 10 min.
The
>pretreatment for both is 15 min microwave in pH 8 EDTA.
>
>Thomas Pier wrote:
>
>>Hello,
>>I am looking for Reinhard von Wasielewski. I recently saw a post
>>stating tha! t he had
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