[Histonet] RE: need some help with frozen sections

[C.M. van der Loos]

   Eva,

   Some DO's and DON'T's with immunostaining of cryostat tissue sections:
     * After  cutting, let the sections dry overnight at room temperature
       under a ventilator
     * Fix  with  cold  acetone (10 min, 4C). Some people apply a "double
       acetone   fixation"  method:  2x  10  min  acetone  fixation  with
       air-drying in between. This may improve the tissue morphology.
     * The  quality  of the acetone should be P.A. grade and don't use it
       twice. Re-distilled acetone cannot be used for fixation of cryo's.
       Traces of water in the acetone ruins the tissue morphology.
     * Be  aware  that  acetone  is not a real fixative like NBF. Acetone
       just  solves  the  fatty  membranes  and  coagulate  the proteins.
       Cryostat  tissue  sections remain quite vulnerable with respect to
       surfactants  like Triton-X100,  Tween-20. These should be avoided.
       Be  aware  that in some autostainer washbuffers surfactants may be
       included.
     * To  solve  this  problem of tissue vulnerability an extra fixation
       (after  acetone-fixation  and  air-drying)  with Zamboni's (1 min,
       RT) is optional. This extra fixation step also improves the tissue
       morphology.  However,  depending on the antigen to be IHC stained,
       this step may also decrease the IHC staining intensity.
     * The  application  of methanol, either as a fixative or as base for
       endogenous   peroxidase   activity  blocking  is (at  least to  my
       vision) absolutely  excluded.  For  example, most human CD markers
       are completely destroyed by methanol. On the other hand I am aware
       of investigators  who  are  using successfully an acetone/methanol
       mixture for fixation of mouse cryo's for staining CD4, CD8 etc.
     * Acetone   is   not   a   good   fixative   when  staining  nuclear
       antigens. This  fixation will  lead to quite fuzzy stained nuclei.
       Instead,  a  NBF-fixation  (5  min,  RT)  works out well. Although
       NBF-fixation is used: do NOT apply heat-induced antigen retrieval.
     * Some  antigens  associated  with  fatty  structures  (for example,
       oxLDL,  or apoB) will solve into acetone. In this case NBF (5 min)
       can be tested. Again, NO antigen retrieval!
     * Endogenous peroxidase activity can be blocked with 0.1% Na-azide +
       0.3%  peroxide in PBS or TBS (20 min, RT). This kills at least the
       endogenous peroxidase activity in erythrocytes effectively, but in
       neutrophils some will remain. If endogenous peroxidase activity of
       neutrophils  is  a  real  problem, glucose oxidase blocking method
       is optional (see Histonet archives).
     * At the end of the IHC staining procedure, after the chromogen step
       wash your slides with tap water. NEVER use distilled water as this
       will ruin the tissue section completely!!!!
     * Unlike  with  FFPE  sections,  aqueous solutions (depending on the
       chromogen  used  of  course)  is  no problem for mounting cryostat
       tissue sections.
     * The  concept  of  first cutting a tissue section and than fixation
       means  "post-fixation".  This  is  something different from a FFPE
       section that  is  first  fixed  as  a block, than embedded and cut
       ("pre-fixation").  This  means that soluble antigens may leak away
       from  a "post-fixed"  tissue  section. Be  aware  of this when IHC
       staining  any small protein (<50KD), cytokine, chemokine, hormone,
       etc.

   I hope this isn't too much frightening you. Happy staining!

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

   phone:  +31 20 5665631
   fax:    +31 20 6960389
   e-mail: [1]c.m.vanderloos <@t> amc.uva.nl




   ----- Original Message -----
                                            From 
   Eva C Anderson <eca9 <@t> georgetown.edu>
                                            Date 
   Mon, 25 Apr 2005 09:59:34 -0400
                                              To 
   histonet <@t> lists.utsouthwestern.edu
                                         Subject 
   [Histonet] need some help with frozen sections

   Good morning,
   I am starting to stain frozen sections for the first time. Until now I
   have  only  stained  paraffin  embedded sections and so was hoping for
   some
   pointers.
   I contacted our histopathology lab who provided me with the following
   information:
   They don't use any antigen retrieval method.
   They  pretreat  the  slides  only  with methanol for 10min followed by
   drying
   before staining.
   Then they proceed with the normal steps of staining and dehydration.
   I have however read that acetone can be used instead of the methanol.
   What  is  the  difference that this provides? I am trying to stain for
   Stat5.
   Is  there  anything  els  I  should  keep in mind when handling frozen
   sections?
   Would  be  very  grateful  for  any information you could provide this
   rookie
   with.
   Thank you,
   Eva

References

   1. mailto:c.m.vanderloos <@t> amc.uva.nl