RE: anti-calretinin and pale brown DAB reaction

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From:"Sebree Linda A." <>
To:'Tibor Ric' <>

Hi Tibor,

As you know, HIER (heat induced epitope retrieval) greatly enhances
calretinin staining.  This can be done a variety of ways in a variety of
buffers; I'm currently using 1mM EDTA, pH 8 for either 2" in a pressure
cooker (Biocare's Decloaking Chamber) or 20" in a 93 degree C water bath.
Both give intense staining using Zymed's polyclonal predilute.  I've also
had success with CellMarque's predilute but had to use a longer HIER

Hope this helps; I'd be happy to try and answer any further questions you
might have.

Linda A. Sebree, HT
University of Wisconsin Hospital & Clinics
Immunohistochemistry/In Situ Hybridization Laboratory
600 Highland Avenue
Madison, WI  53792-2472

FAX: (608)263-1568

> -----Original Message-----
> From:	Tibor Ric []
> Sent:	Wednesday, April 12, 2000 12:49 PM
> To:	HistoNet Server
> Subject:	anti-calretinin and pale brown DAB reaction
> Hi everybody!
> I'm using anti- calretinin from Swant (Code No 7699/4, polyclonal) in my
> project. The problem is that I'm getting too pale DAB reaction. I did
> everything like before for HBME-1 and CK 5/6 and they were not that pale
> brown. I'm sure that the DAB solution is okay. The material I'm using is
> from 1991 to 1999.
> One problem I consider here would be the fixation time which I don't know
> for those older blocks.
> Please, come with suggestions and good ideas for a beginner...
> Today we tried a new antibody from Novocastra Laboratories (clone 5A5,
> monoclonal) and I'm still having that pale brown color...
> The positive controls were pale too.
> Thanks in advance!
> tibor ric
> a Swedish student from the University of Karlstad, Sweden

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