RE: anti-calretinin and pale brown DAB reaction
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From: | "Sebree Linda A." <la.sebree@hosp.wisc.edu> |
To: | 'Tibor Ric' <tibor@home.se> |
Reply-To: | |
Content-Type: | text/plain |
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
procedure.
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
D4/218-2472
600 Highland Avenue
Madison, WI 53792-2472
(608)265-6596
FAX: (608)263-1568
> -----Original Message-----
> From: Tibor Ric [SMTP:tibor@home.se]
> 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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