[Histonet] IHC staining of previously H&E-stained prostate cores

Thomas Jasper tjasper <@t> copc.net
Mon Jun 23 19:22:41 CDT 2008

Just a thought...it might be the fact that the plastic coverslip is
removed by soaking in acetone (your only choice).  I know that we've had
a similar experience here.  The situation - our previous prostate needle
double stain (p504s and HMW-CK) used to have to be done as 2 separate
runs.  This is no longer the case, however, when it was, folks would
occasionally forget to hold over the slides that were to receive the
next stain.  These of course would then be coverslipped.  We would
remove the coverslips (acetone) and run them back to water, then rerun
for the second Ab.  Every time this happened the 2nd Ab never worked.
By process of elimination, we've deduced that the acetone exposure was
the limiting factor.  I'm not a chemical engineer, but I believe that
was our problem and it may be your problem as well.  I'm thinking that
the acetone does something to the binding sites rendering them unable to
take either the Ab.  Or maybe they can take the Ab but the chromagen
step is compromised by the acetone.  Either way it wouldn't work, we
stopped doing it and our staining was fine.  I suspect this problem
would happen with any Ab and probably any chromagen.  Any chemistry
gurus out there could weigh in, but I don't think you'll be able to
stain those biopsies.  Hopefully you've got extras cut that are copies
of the H and E?  Maybe not as you've been asked to run IHC on them.  I
know this does not solve the problem, but maybe it explains why you're
having it.
Good luck,
Tom J.

Thomas Jasper HT (ASCP) BAS
Histology Supervisor
Central Oregon Regional Pathology Services
Bend, Oregon 97701
tjasper <@t> copc.net 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
JMyers1 <@t> aol.com
Sent: Monday, June 23, 2008 8:04 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] IHC staining of previously H&E-stained prostate

I'm trying to stain previously H&E-stained prostate core biopsies  for
HMW-CK and P504S, and I've been obtaining less than ideal results.  I
suspect that this might be caused by inadequate removal of mounting
media (once the tape-coverslip is removed with acetone) or over/under-
retrieval, but I could really use some advice from someone who has
already  worked through a situation like this.

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