[Histonet] RE: IHC double-labeling -- same secondary?

From:"C.M. van der Loos"

   Hi Anna Elisse,

   To  my  opinion  there  is  a  strong  preference of using second step
   reagents  of  the  same host. That makes it very safe to mix them in a
   cocktail   (time   saving!)   without   any   chance   of   unexpected
   cross-reactions.  In  your  case  you could mix donkey anti-rat/FLUO +
   donkey   anti-goat/biotin.  Don't  forget  that  you  could  mix  your
   primaries in a cocktail as well!

   Good luck,

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

   Date: Wed, 29 Mar 2006 10:52:59 -0500 (EST)
   From: "Anna Elisse Beaudin" 
   Subject: [Histonet] IHC double-labeling -- same secondary?
   To: Histonet@lists.utsouthwestern.edu
   Hi all,
     I am performing double labeling IHC on mouse brain tissue with two
   primaries  from  different  species,  one  raised in rat, the other in
   I was wondering if it was kosher (or even preferred) to use both a
   donkey  anti-rat  secondary  and a donkey-anti goat secondary.  if so,
   I incubate with both together as a cocktail?  my donkey anti-rat is
   flourophore conjugated, and I was planning to use biotinylated donkey
   anti-goat followed with a flourophore-conjugated streptavidin.  any
   suggestions/comments would be greatly appreciated.
   Thanks in advance,
   Anna Beaudin
   Division of Nutritional Sciences
   Cornell University
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