[Histonet] RE: Antibody staining on non-pfa fixed tissue
C.M. van der Loos
c.m.vanderloos <@t> amc.uva.nl
Mon Jul 31 02:56:27 CDT 2006
Dear Greg,
As you already concluded the epitope of interest is PFA-sensitive. How
about using a non-crosslinking fixative here? For example methacarn
(methanol:chloroform:acetic aced = 6:3:1) does not contain
formaldehydes. Fix your sample for 48 hours, dehydrate via alcohol
series and embed in paraffin. Because methacarn is non-crosslinking
fixative most antigens doesn't need antigen retrieval.
Just a suggestion...
Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
Date: Fri, 28 Jul 2006 18:46:26 +0200
From: "Gregory A. O'Sullivan" <osullivan <@t> mpih-frankfurt.mpg.de>
Subject: [Histonet] Antibody staining on non-pfa fixed tissue
To: "HistoNet" <histonet <@t> lists.utsouthwestern.edu>
Hello HistoNetters,
I realise that this may be a question that has arisen on previous
occasions, however, I have been unable
to find an exact answer from a search of previous email correspondence
on HistoNet.
I would like to detect and examine the distribution of my protein of
interest in mouse brain sections
(with particular emphasis on the hippocampus) and compare it to other
known markers.
The problem is that the antibody I am using is extremely sensitive to
pfa fixation but the marker antigens
are best detected on fixed tissue. I have tried to overcome this
problem by fixing cryostat sections
briefly with pfa before using different antigen retrieval protocols
(either microwave or heated bath incubatio! n
in C
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