Re: IHC on Glutaraldehyde Fixed Tissues

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From:"R.Wadley" <>
Date:Wed, 09 Jun 1999 08:57:51 +1000
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	Dear Paul,

	The problem with glutaraldehyde is that the bonds it forms during fixation
are permenent.  ie fomaldehyde creates single bonds which can be reversed,
but gutaraldehyde creates double bonds which tend to stay bound.  For
immuno EM you will note that most methods use a
paraformaldehyde/glutaraldehyde mix where the glutaraldehyde concentration
is quite low (1-2%) & fixation times are not excessive.
	If your tissue has been fixed for routine (non-immuno) EM then you will
have problems.  The longer the tissues were stored in glutaraldehyde the
greater your problems are likely to be.  More agressive antigen retrieval
(perhaps enzyme digestion) may help, but you may run the risk of damaging
those antigen sites that are already available.
	If your tissue has been post fixed in OsO4 then you have virtually no hope
of success.
	If this work is going to be ongoing, get your fresh sample fixed as for
Immuno EM, or do your initial fixation in formaldehyde then send a bit off
to EM for them to refix as per their routine protocols.  

	Rob W.

At 06:11 PM 6/8/99 -0400, you wrote:
>I have tissues which were fixed in glutaraldehyde and then embedded in 
>Does anyone have experience in performing IHC on sections processed this
>Routine methods (antigen retrieval with citrate buffer etc.) do not seem to 
>produce good results.
>The tissue were originally designated for EM. 

R. Wadley, B.App.Sc, M.L.S
Laboratory Manager
Cellular Analysis Facility
School of Microbiology & Immunology
UNSW, New South Wales, Australia, 2052
Ph (BH) 	+61 (2) 9385 3517
Ph (AH)	+61 (2) 9555 1239
Fax 	+61 (2) 9385 1591
	(Under development)

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