[Histonet] RE: Can Alk phs be run on peroxidase slides?

[C.M. van der Loos]

   Dana,

   Basically  I  think  this  is  possible.  In  the  end  it's a kind of
   sequential double staining. Some remarks:
     * Of course you have to be aware of animal species: if your AP stain
       is  another  mouse  primary you may end up with a cross-reactions.
       This  type  of cross-reaction is not really expected since DAB was
       used   as   chromogen. DAB   reaction   product   has  the  unique
       characteristic of effectively 'sheltering' all immunoreagents used
       in the first staining sequence.
     * If  you  want to be on the safe side with cross-reactions, perform
       HIER (using the same buffer as for the first one) for just 5 min +
       10  min cooldown. This effectively removes or at least destroy all
       your  first  step  reagents while the DAB reaction product remains
       unaffected.
     * A  second streptavidin-biotin detection system is not recommended.
       To  be  on  the  safe  side  use a AP-labeled polymer. In case you
       really  need another streptavidin-biotin detection system, perform
       a  biotin blocking first (avidin 0.1%, 15 min - d-biotin 0.01%, 15
       min, or a commercial biotin blocking kit).
     * Given for example that your first DAB stain is cytoplasmic and the
       second  AP  stain  is nuclear, AP in red with Liquid Permanent Red
       (Dako) is a good choice.
     * Destaining  of a DAB-stained slide will be a very hard job, not to
       say impossible.

   Lots of success,

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


   Date: Mon, 24 Jul 2006 12:44:48 -0400
   From: "Dana Settembre" <settembr <@t> umdnj.edu>
   Subject: [Histonet] Can Alk phs be run on peroxidase slides?
   To: <histonet <@t> pathology.swmed.edu>
   Hi,
   One  of  my  pathologists  is  asking  if  I can take a slide that has
   already
   been stained with a
   peroxidase/avidin/biotin/DAB detection system and re-stain with an
   Alkaline Phosphotase detection system.
   Can it work?
   Must it be de-stained?
   Any info will help.  Thanks,
   Dana Settembre
   Immunohistochemistry Lab
   University Hospital - UMDNJ
   Newark, NJ