Hi Tim,
I've been doing immunos close to 20 years and did not encounter this problem
until HIER came into play. It has something to do with heat retrieval with
particular antibodies ( I have a problem with
Vimentin and cytokeratin, clone 34 BE12). If anyone has any suggestions for
reason and possible remedy, I'm sure we'd all be grateful.

Katri

----- Original Message -----
From: "Coskran, Timothy M" 
To: 
Sent: Thursday, December 12, 2002 2:29 PM
Subject: IHC


> Hello,
>
> Has anyone ever encountered the following type of IHC background problem:
> With two separate antibodies, one a cytoplasmic marker and the other a
> membrane marker, every single nucleus in the section turns brown.  The
stain
> itself works, as most of the time you can read through the background, but
> all test slides and negative controls (omission of primary) are affected.
>
> Both antibodies are rabbit polyclonals, both are steam retrieved one with
> citrate pH 6 the other with EDTA.  Both are detected using a whole IgG
> biotinylated goat anti rabbit secondary followed by Elite ABC and Dako
> Liquid DAB+.  Blocks included 3.0% hydrogen peroxide (aq), Dako Biotin
> Blocking system and Dako Protein block.  All were stained on an IHC
stainer.
>
> In addition, both of theses antibodies have been reliable for months
(using
> the above protocol steps), until now......
>
> Thanks,
> Tim Coskran
> Pfizer
>
>
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