[Histonet] Frozen section problems!!

From:Adam M Gomez

   We  are  wanting  to  section  peripheral  gang=  lia  at 10um using a
   cryostat. Our current procedure is as follows:
   1)perfused with PBS/8% Para, ganglia tissue removed
   <= FONT face="Default Sans Serif,Verdana,Arial,Helvetica,sans-serif"
   color=  =#000000 size=4>2)tissue rinsed in PBS under agitation for
   10 min.
   3)placed in 30% sucrose overnight (or until= tissue sinks)
   4)rinsed in PBS/dH20
   5)embedded in OCT
   6)frozen rapidly in liquid nitrogen
   7)tissue  allowed  to  equilibrate  to  cryostat temp & sectio= ned in
   cryostat (-18 C)
   8)sections= are picked up with room temp slides and dried at RT for an
   9)Slides  are  dipped  in clearing agent (xylenes)= followed by graded
   alcohol  (95%, 70%, 50%), dH2O, cresyl violet stain, dH2= 0, 50%, 70%,
   95%, & xylenes...and coverslipped immediately.

   The problem we are having is that the tissue looks outstanding after    sectioning  as  long  as  it remains wet (i.e., when we take a look at
   tissue  d= uring the cresyl violet stage above)--once it begins to dry
   we're losing ce= lls and overall tissue integrity declines rapidly--It
   seems  that  the  dehydr=  ation  steps  following the staining of the
   tissue  is  the culprit. We have tr= ied several modifications of this
   procedure but have yet to find a successf= ul way to prevent damage of
   the  tissue.   If  you have any suggestions p= lease let me know. This
   problem has been detrimental to our work for the pa= st 3 months.


      Adam Gomez
   Research&nb= sp;Technician/Assistant
   University of Nebraska at Om= aha
   Department of Psychology
   (402) 554-6028
   = [1]amgomez@mail.unomaha.edu


   1. 3D"mailto:amgomez@mail.unomaha.edu"
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