Hi David,

Our cytology department routinely fixes any cytology preparations destined
for IHC in acetone for 3.5 minutes. This seems to work for most immuno
stains.  Several exceptions are ER & PR; these seem to need special
attention such as fixation in Zamboni's fixative or the 3-step 10%
NBF/methanol/acetone fixation however we still haven't found a reproducible
fixation protocol. On at least one occasion we have achieved positive
staining of acetone fixed cytospins for ER and PR but again we have not been
able to reproduce this.  One other exception is Calretinin; although
fixation is the same acetone protocol, we do a heat pretreatment of 2" in
the Biocare DeCloaking chamber with Biocare's Nuclear DeCloaker buffer or
20" in a 93 degree C waterbath in the same buffer.  Dana Dittus suggested
heat pretreatment and explained to me the mechanism involved (its not really
the same antigen retrieval mechanism one usually associates with FFPE
tissues) but she would be better able to explain this than I.

Our cytopathologists are more and more going with cell blocks when there is
enough material to make one and this suits us just fine as we know how to
treat these.

Linda A. Sebree, HT(ASCP)
University of Wisconsin Hospital & Clinics
IHC/ISH Laboratory
A4/204-2472
600 Highland Ave.
Madison, WI 53792-2472
(608)265-6596
FAX: (608)262-7174


-----Original Message-----
From: David Grehan [mailto:david.grehan@olhsc.ie]
Sent: Wednesday, October 24, 2001 5:50 PM
To: 'histonet'
Subject: fixation procedures for cytology immunocytochem


Hi histocybernetters
I would like to get your opinions on procedures for cytology specimens
that are prepared for  immunocytochemistry. I am currently fixing direct
smears or cytospins immediately in 95% absolute alcohol for 10 mins .I
do this for any cytoplasmic or cell membrane antigens . I fix in
methanol:acetone 50:50 for 10 mins for any nuclear antigens such as Tdt
or Myo D1 (to increase permeabilisation)is this necessary?. I work in a
busy paediatric laboratory so we don't get a lot of cytology specimens
but on occasion we are requested to do them. My results to date have
been patchy especially following storage at -20C. I would like to be a
bit more confident about my procedures and would like  any comments from
anyone doing a lot of immunocytochemistry on cytologyy specimens . Also
how do you store your smears ? Do you spray fix or use PEG.?