Tim,

I've encountered background staining in CK903 (34BE12) in slides cut for me
by routine histology.  The background was in a definite line to where the
section was being picked up off the water bath.  When I cut slides for this
one, the background wasn't there.  Solution:  this antibody is sensitive to
the adhesive routine histology was using in there water baths.  Sta-on from
Surgipath is the best adhesive around but 34BE12 likes to recognise it as a
high molecular weight cytokeratin.  This is the only antibody that I found
picked up on that adhesive (I don't use adhesive in my waterbath).  As for
vimentin, it is so ubiquitous that it could possibly be an adhesive problem
as well.

Good Luck,

Glen Dawson

-----Original Message-----
From: Katri Tuomala [mailto:katri@cogeco.ca]
Sent: Saturday, December 14, 2002 1:59 PM
To: Coskran, Timothy M; Histonet@pathology.swmed.edu
Subject: Re: IHC


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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