Re: IHC on Glutaraldehyde Fixed Tissues

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From:Barry Rittman <>
Date:Wed, 09 Jun 1999 06:54:41 -0700
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		I agree entirely with the comments by Rob. Glutaraldehyde is difficult if
not impossible to remove for IHC. You are better off avoiding
glutaraldehyde for IHC. The main problem is that glutaraldehyde not only
binds tenaciously but some unbound will remain in tissues and cause
excessive background staining.
Some attempts we made at neutralizing aldehyde groups present were
partially successful but gave variable results with the same tissue and
Good luck

At 08:57 AM 6/9/99 +1000, you wrote:
>	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.  
>	Regards
>	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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