I have seen nuclear staining when it is not supposed to be there in cases
that are over heated with HIER or over digested with EIER, no explanation
for it, but it does indicate to me that I should cut back on the heating or
digestion until it goes away.
Patsy


----- Original Message -----
From: "Katri Tuomala" 
To: "Coskran, Timothy M" ;

Sent: Saturday, December 14, 2002 12:58 PM
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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