[Histonet] Re: Histonet Digest, Vol 15, Issue 39

[John Auld]

Hi Jean

34BE12 is notorious for inconsistency, I have previously used it with a
mixture of Heat and trypsin (20 minutes MW in Citrate @ pH 6.0 followed by
15 seconds trypsin be careful not to overdo the trypsin as it can
overdigest in a very few seconds. Cytokeratin 5/6 has been found to give
more consistant results.

Hope this is of some help

John

John Auld MSc CSci FIBMS
BMS 4
Dept of Histopathology and Clinical Cytology
Arrowe Park Hospital
Arrowe Park Road
Upton
Wirral

External Tel 0151 604 7025
Message: 13
Date: Fri, 25 Feb 2005 11:02:57 -0000
From: "Jean Gillson" <jean.gillson <@t> elht.nhs.uk>
Subject: [Histonet] 34BE12 on prostate[Scanned]
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:

<1030B679AD69D6119C3F00080210DD9D05AC0348 <@t> bhrv-nt-11.bhrv.nwest.nhs.uk>

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Dear all fellow histologist,

We seem to have a recurrent problem with 34BE12 IHC staining on both
prostate chippings and prostatectomy specimens.  Some basal cells stain
whilst others that should stain are negative.  Our procedure is Trypsin for
12 minute at pH 7.8 and 37 degrees C.  We use a Dako autostainer machine
and
our antibody is from Dako.  Previous spec sheets state Trypsinisation but
our most recent one states heat retrieval.  So as a test we did both
retrieval methods.  Oddly enough, microwaved in Dako retrieval solution was
slightly better in prostatectomy specimens than trypsin (still uneven
staining) and worst in chipping specimens.  Can anyone offer any advice?
What antibody panels are people doing for prostate cases?  Help desperately
needed!!

Jean Gillson BMS2

Histology Department

Blackburn Royal Infirmary

Blackburn

UK