Hi,

   I  have  had  this  experience before. I am also staining the cytospin
   slide  and  I  am using the HeLa cells as my control. It took me quite
   some time to troubleshoot the problem. In my case, the problem lies at
   the    fixative    that    I've   used.   At   first   I   was   using
   4%paraformaldehyde/PBS.  This  fixative  is  really not good at all at
   preserving  my  protein  of  interest. The protein lasted for only one
   day.  I  didn't  know that because I did my staining on the day itself
   after  cystospining  and fixing  the cells. Later on, when I did again
   the  staining  2-3 days, I couldnt get back my positive signal. Then I
   decide  to  change  the fixative, I think 10% NBF is the best but this
   aldehyde  gives  very high background in fluorescence staining. I have
   also  tried  alcohol-based fixative and found that absolute ethanol is
   the  best. Just love it! It gives me very low (almost none) background
   and  preserves  my  protein  very  well.  In  my case, I am totally in
   agreement with Terry on methanol.

   I  would  suggest  that  you  try out different fixatives to see which
   one is suitable for your protein.
   With regards,
   TIANG YEE PENG
   Department of Medical Microbiology,
   Faculty of Medicine,
   University of Malaya.
     _________________________________________________________________

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References

   1. http://g.msn.com/8HMAENUS/2737??PS=47575
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