[Histonet] RE: IHC cytology specimens

From:"C.M. van der Loos"

   Mary Ann,

   I  am answering this from a research point of view, totally unaware of
   Her2neu guidelines or what so ever.

   We  fixed  cytospins in NBF (5 min, RT), washed them with tap water (5
   min) and then to alcohol 70 (3 hours - overnight). Wash with tap water
   and perform HIER with appropriate buffer. We use half the time at max.
   temperature as for FFPE's. After cooling down, wash with tap water and
   perform   IHC  as  with  FFPE  tissue  sections  (antibody,  dilution,

   I  am  aware  this  procedure  as  against  all logics but  it  simple
   works. After  all,  5  min NBF fixation cannot cause any cross linking
   and  alcohol  fixation  certainly  not. Therefore,  HIER  would not be
   necessary.  But  after  testing we saw HIER was very essential! When I
   heard  about  this  procedure  I  was  very  sceptical  it  would ever
   work. However,  I was surprised to see how well the cells survive this
   harsh  procedure  and  nicely  stay  at the glass. For some particular
   antigens we  have  even done this procedure successfully with cryostat
   tissue  sections from fresh frozen blocks (section stays at the glass,
   morphology is fine)!

   Therefore  folks:  a  theory  is  good to keep in mind, but testing is
   sometimes  better.  And,  I  also stopped with surprising myself about
   "strange" protocols.

   I hope this helps.

   Have a nice weekend,

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands

   Date: Thu, 24 May 2007 11:16:19 -0500
   From: "Deathridge, Mary Ann" 
   Subject: [Histonet] IHC cytology specimens
   Does  anyone  in  histoland  have  a protocol for the prep of cytology
   specimens (cytospins, smears) for IHC?
   Formalin  fix?  how  long? air dried? 95% alc fix?  esp. since the new
   Her2nu guidelines
   Thanks in advance

   MaryAnn Deathridge, HT (ASCP)
   Supervisor, Histopathology
   Vanderbilt Un. Med. Ctr
   Nashville, TN
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