Dana, I don't have your work e-mail address so I'm trying to find you
through Histonet.  Since I referenced you concerning heat pretreatment for
calretinin on cytospins, I've had several requests for your input (see
below).  Would you care to explain (through Histonet) the mechanism as you
once did for me via phone?

Thanks and sorry to put you on the spot,

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: Laurie Brett [mailto:lauriebrett@bigfoot.com]
Sent: Friday, October 26, 2001 12:32 PM
To: Sebree Linda A.
Subject: Re: fixation procedures for cytology immunocytochem


Interesting, we've also found that heat pre-treatment does the business for
ER and PR although fixation is in Acetone/Methanol (50/50) so there
shouldn't be any cross linking. Would Dana Dittus care to explain the
mechanism to the rest of us?

Lawrence Brett
Immunocytochemistry Lab.
Pathology Dept.
Western General Hospital
Edinburgh  EH4 2XU
Tel: 0131 537 2148
email: lauriebrett@bigfoot.com

> From: "Sebree Linda A." 
> Date: Thu, 25 Oct 2001 08:23:07 -0500
> To: 'David Grehan' 
> Cc: 'Histonet' 
> Subject: RE: fixation procedures for cytology immunocytochem
> 
> 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.?
> 
>