[Histonet] RE: Double immunostaining with a rabbit and rat

[C.M. van der Loos]

   Hi Cathy,

   I  cannot  recommend you this G/2 kit as this is sequential as well as
   multi-species based. A home-brew solution is more appropriate here:

   Rt CD34 + Rb casp-3 (cocktail)

   Goat  anti-rat/HRP  (mouse absorbed)* + goat anti-rabbit/AP polymer **
   (cocktail)

   Develop AP activity in red (Dako LPR)

   Develop HRP activity in brown (DAB+ from any source)

   * =  see Jackson or Rockland

   ** = see LabVision

   Lots of success with staining!

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


   Date: Mon, 02 Apr 2007 14:40:59 +1000
   From: Cathy Malcontenti-Wilson <cmalc <@t> unimelb.edu.au>
   Subject: [Histonet] Double immunostaining with a rabbit and rat
   primary antibody
   To: histonet <@t> lists.utsouthwestern.edu
   Dear All,
   We wish to do a double immunostain for CD34 and active caspase 3 on
   paraformaldehyde   fixed  mouse  tissues.  The  cd34  primary  is  rat
   monoclonal
   anti cd34  The caspase 3 primary is a rabbit polyclonal antibody.
   We  would  like  to use the Dako Envision G/2 kit for double staining,
   however
   this  kit  is for mouse or rabbit primaries only. When we  do a single
   stain
   for  cd34,  we use a Rabbit anti Rat IgG as a linking antibody between
   the
   primary and secondary antibody (Dako Envision plus kit for rabbit
   primaries) and this works well on our tissues in our experience.
   My question is: Can I add this linking antibody step to the method and
   still use the Envision G/2 kit?
   Does anyone have ! any exper