[Histonet] RE: Elution buffer on frozen tissues

[C.M. van der Loos]

   Dear Sun Zhon,

   You  better  don't  use  an elution buffer on cryo's during sequential
   double  staining  at  all.  In  fact  you  don't need it and with high
   affinity primaries it's not even effective. When performing sequential
   doublestaining   one MUST  apply DAB  as  chromogen  after  the  first
   staining sequence. DAB is the only enzymatic reaction product that has
   the   unique   characteristic   of   sheltering   the   first  set  of
   immunoreagents      effectively.      This      DAB-layer     prevents
   cross-reaction with the second staining sequence.

   There  is  one important thing you should take care of: an appropriate
   dilution   of   the  first  step  primary  antibody. If  this  is  too
   concentrated,  the  DAB  sheltering effect is not completely effective
   and cross-reaction with the second staining sequence will occur.

   Good luck!

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



   Date: Mon, 3 Oct 2005 16:35:32 -0700 (PDT)
   From: Sun Zhon <ihctech2000 <@t> yahoo.com>
   Subject: [Histonet] Elution buffer on frozen tissues
   To: histonet <@t> lists.utsouthwestern.edu
   Hi All,
   I  am  going  to run a dual chromogen staining on frozen tissues. I am
   wondering  whether  I  can  use  the  elution  buffer to elute the 1st
   antibody  complex?  Will  the elution buffer destroy morphology of the
   frozen tissues?
   Thanks in advance for the help.