[Histonet] Double immunostaining with a rabbit and rat primary antibody

Cathy Malcontenti-Wilson cmalc <@t> unimelb.edu.au
Sun Apr 1 23:40:59 CDT 2007

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 experience with this sort of thing or any different 
ideas on doing double staining using these particular antibodies?
All ideas and comment appreciated as always.

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