[Histonet] antigen retrieval for IHC

[Bryan Hewlett]

Patsy,

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.

Best regards,

Bryan

----- Original Message ----- 
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
To: <jkiernan <@t> uwo.ca>; "'Maria Mejia'" <maria <@t> ski.org>
Cc: <histonet <@t> lists.utsouthwestern.edu>
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 
> 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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