Re: Antigen Retrieval

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From:"Genty Family" <> (by way of histonet)
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Hi Catherine:

It has been my experience that Vimentin is actually enhanced by Heat induced
AR with 0.01M citrate buffer pH 6.0.  However I do not know the effects of
the protease and the HCl on Vimentin antigenicity.  Perharps some of our
histonet gurus can provide some insight.

When performing Brdu stains, I used to use a similar procedure, AR with
0.01M citrate buffer followed by an 60 min  RTemp incubation in 2N HCl then
by a standard HRP-labeled streptavidin technique.  2N HCL times may be lower
these days.

You may want to try this, in a worst case scenario the antigenicity may be
affected by the HCl.  But even if that were the case you could reverse the
order and adjust your titers and detection systems accordingly.

I have not kept up with the literature, but there may be even simpler
approaches to Brdu IHC that may better preserve your Vimentin antigenicity.

I hope this is somewhat helpful.

Carlos Genty
Breast Cancer Center
Baylor College of Medicine
Houston, Texas
----- Original Message -----
From: Bennett, Catherine (Katie) <>
To: 'Histonet Server' <>
Sent: Thursday, December 23, 1999 10:51 AM
Subject: Antigen Retrieval

> I realize not many histonetters may be online right now, but to those out
> there, I present my current dilemma.
> I am trying to do double immuno staining for BrdU and Vimentin in 10%
> neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> labeling with almost all of the microwave antigen retrieval procedures
> been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> EDTA).  But the Vimentin stain after microwave AR is really weak.  Without
> antigen retrieval, I get no BrdU staining but awesome Vimentin stains.  My
> protocol always includes a 5 minute 0.05% Protease K enzyme digestion
> and a 1h HCL denaturation step.  Without the microwave AR, these alone
> do the trick for the BrdU.
> I'm guessing my next step is to ditch the microwave AR since it kills the
> vimentin and play around with various enzyme digestion times to boost the
> BrdU.
> Anyone have any insight or suggestions?
> ****************************
> Catherine "Katie" Bresee Bennett
> Sr. Technical Associate
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico

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