[Histonet] antigen retrieval for IHC

[Patsy Ruegg]

           Shi mentions in his book on pg.10 ANTIGEN RESTORATION "Simply
bathing deparaffinized sections in a cold 20% sucrose-saline solution could,
over several days, restore a certain amount of immunoreactivity."

In Introduction to IHC by Polak and Van Noorden on pg 24 3.8.1 "The simplest
form of reversing the effects of formalin is to wash the tissue well before
processing"

I am about to test these statements, as I just received tissues for an IHC
project that have been in 10% NBF for over one year.  I washed the tissues
in running tap h20 for 2 hrs., I will now process them into paraffin.  I am
planning as well to put some of the sections in 20% sucrose at 4dc for 2
days if I have trouble getting good IHC signal.  I will keep you all posted
on this experiment.  These are samples of mouse mammory tissue.  I will be
testing them with cleaved caspase 3 and Ki67 to start.  I have both of these
antibodies worked out very well for ffpe tissues fixed 24-48 hrs.  

Patsy


Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pruegg <@t> ihctech.net
web site www.ihctech.net
 

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-----Original Message-----
From: John Kiernan [mailto:jkiernan <@t> uwo.ca] 
Sent: Sunday, September 25, 2005 11:14 PM
To: Maria Mejia
Cc: pruegg <@t> ihctech.net; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] antigen retrieval for IHC

Maria, can you give chapter and verse for the Shi/Taylor and Puchrler/Meloan
papers? Most papers with Shi and Taylor among the authors are about high
temperature antigen retrieval (boiling water, with pH optima for various
antigens).
 
Holde Puchtler and Susan Meloan published many imortant papers about
staining ad histochemistry in the 1970s=1980s. Susan M was a histonetter in
the 1990s. 

*****************
 
Maria Mejia wrote:
> 
> Hello,
> 
> I believe it was sometime in June of this year that there was a 
> discussion on the histonet regarding "the simplest form" of reversing 
> the effects for formalin fixation on tissue was to wash the tissue 
> well (before) processing.
> 
> Well, I just read the article "Antigen retrieval IHC: Past, Present, & 
> Future by Shan-Rong Shi, richard J. Cote & Clive R. Taylor (can google 
> this article). It's a very interesting! Anyway, the article has a 
> section titled non-heating AR method stating (this) same simple method 
> by (Puchtler & Meloan). It also goes on to say that Elias JM (1990) 
> adopted this method routinely by storing deparaffinized in fresh 
> changes of 10% sucrose/PBS @ 4C overnight (before) IHC.
> 
> My questions are, does anyone know or tried this method used by Elias?
> And, can anyone explain the mechanism of 10% sucrose/PBS play in this 
> AR method?
> 
> Just curious!
> 
> Maria Bartola Mejia
> Smith-Kettlewell Eye Research Institute San Francisco, CA 94115
> Email: maria <@t> ski.org
> Phone: (415)-345-2185
> 
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