Dear Yves,

   What  Gayle  Callis  already  mentioned  about  avidin-biotin blocking
   before  the first and the second sequence is indeed the only thing you
   can  do  when  combining  two  biotin  techniques  in double staining.
   However,  in  general I don't recommend this for multiple staining. To
   my  opinion there is still a great risk for false positive results. As
   Gayle  described,  the multistep procedure combining one unlabeled and
   one  biotinylated  antibody works fine. I would like to add the option
   of  biotinylating or the labeling with an Alexa fluorochrome of one of
   your primaries using the Zenon kit.

   Good luck!

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands
   phone:  +31 20 5665631

   Date: Tue, 14 Mar 2006 14:13:49 -0700
   From: Gayle Callis 
   Subject: Re: [Histonet] double/triple staining with streptavidin-Alexa
   Fluor
   To: Yves Heremans ,
   Histonet@lists.utsouthwestern.edu
   Yes,  however,  using  an avidin/biotin blocks before each antibody to
   ensure
   1)  quenching  endog  biotin  before  first and quenching any residual
   biotin
   left  over  from  first  antibody  sequence  before  you do the second
   primary
   sequence.
   We do double IFA staining with two rat antimouse monoclonals in the
   following  way  with  one  purified  monoclonal  and  one biotinylated
   monoclonal
   primary.
   Frozen  sections  fixed  with  acetone  alcohol  mixture  at  RT after
   overnight drying
   Normal serum block matched to host of secondary used
   Strepavidin/biotin block (Vector)
   Rat antiMouse monoclonal
   Donkey antiRat F(ab')2 frag of IgG conjugated to RRX
   Rat serum block, 5% for 15 minutes
   Pri! mary #2 r
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