[Histonet] RE: Antibody staining on non-pfa fixed tissue

[C.M. van der Loos]

   Dear Greg,

   As you already concluded the epitope of interest is PFA-sensitive. How
   about  using  a  non-crosslinking fixative here? For example methacarn
   (methanol:chloroform:acetic    aced    =   6:3:1) does   not   contain
   formaldehydes.  Fix  your  sample  for 48 hours, dehydrate via alcohol
   series  and  embed  in paraffin. Because methacarn is non-crosslinking
   fixative most antigens doesn't need antigen retrieval.

   Just a suggestion...

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


   Date: Fri, 28 Jul 2006 18:46:26 +0200
   From: "Gregory A. O'Sullivan" <osullivan <@t> mpih-frankfurt.mpg.de>
   Subject: [Histonet] Antibody staining on non-pfa fixed tissue
   To: "HistoNet" <histonet <@t> lists.utsouthwestern.edu>
   Hello HistoNetters,
   I  realise  that  this  may  be a question that has arisen on previous
   occasions, however, I have been unable
   to find an exact answer from a search of previous email correspondence
   on HistoNet.
   I  would  like to detect and examine the distribution of my protein of
   interest in mouse brain sections
   (with  particular emphasis on the hippocampus) and compare it to other
   known markers.
   The  problem is that the antibody I am using is extremely sensitive to
   pfa fixation but the marker antigens
   are  best  detected  on  fixed  tissue.  I have tried to overcome this
   problem by fixing cryostat sections
   briefly  with  pfa  before using different antigen retrieval protocols
   (either microwave or heated bath incubatio! n
   in C