Re: [Histonet] antigen retrieval for IHC
You will need to wash MUCH longer than 2 hours in running tap water in order
to obtain significant reversal after I year in NBF.
The Helander paper quoted in my NSH handout gives about 6 days to effect a
90% reversal and that was following 24 hour fixation.
----- Original Message -----
From: "Patsy Ruegg"
To: ; "'Maria Mejia'"
Sent: Monday, September 26, 2005 11:58 AM
Subject: RE: [Histonet] antigen retrieval for IHC
> Shi mentions in his book on pg.10 ANTIGEN RESTORATION "Simply
> bathing deparaffinized sections in a cold 20% sucrose-saline solution
> 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
> form of reversing the effects of formalin is to wash the tissue well
> 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
> 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
> 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
> antibodies worked out very well for ffpe tissues fixed 24-48 hrs.
> Patsy Ruegg, HT(ASCP)QIHC
> IHCtech, LLC
> Fitzsimmons BioScience Park
> 12635 Montview Blvd. Suite 216
> Aurora, CO 80010
> wk email email@example.com
> web site www.ihctech.net
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> -----Original Message-----
> From: John Kiernan [mailto:firstname.lastname@example.org]
> Sent: Sunday, September 25, 2005 11:14 PM
> To: Maria Mejia
> Cc: email@example.com; firstname.lastname@example.org
> Subject: Re: [Histonet] antigen retrieval for IHC
> Maria, can you give chapter and verse for the Shi/Taylor and
> papers? Most papers with Shi and Taylor among the authors are about high
> temperature antigen retrieval (boiling water, with pH optima for various
> Holde Puchtler and Susan Meloan published many imortant papers about
> staining ad histochemistry in the 1970s=1980s. Susan M was a histonetter
> the 1990s.
> Maria Mejia wrote:
>> 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: email@example.com
>> Phone: (415)-345-2185
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