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

Tarango, Mark mtarango <@t> nvcancer.org
Mon Apr 2 19:50:31 CDT 2007

NO!  Don't use the rabbit link.  You need a mouse link.  I'm pretty sure
that kit works by having two different primary antibodies in DIFFERENT
species (one in MOUSE and one in RABBIT). You already are using a rabbit
primary for the caspase 3, so you need mouse now. 

If you want to link it up so the Dako kit that you have, will pick up
the signal from the rat primary, then you need to turn that rat into a

Use a mouse anti-rat antibody (doesn't have to conjugated to anything).
Then you end up with the caspase 3 being rabbit and the CD34 being
mouse.  When you do this, you end up amplifying the signal, you might
want to keep that in mind, but it probably doesn't matter.  You just
want to know if the cells are positive I'm guessing, since you're using
polyclonal stuff anyway (polyclonals binding to various random epitopes
would be tough to quantify, but you can find out if there is antigen
being expressed).

Make sure that the antibody you buy is highly cross-absorbed against
other species...

Mark Adam Tarango HT(ASCP)

Histology/Immunohistochemistry Supervisor

Nevada Cancer Institute

One Breakthrough Way

Las Vegas, NV  89135

mtarango <@t> nvcancer.org

Direct Line (702) 822-5112

Fax (702) 939-7663


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Cathy
Sent: Sunday, April 01, 2007 9:41 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Double immunostaining with a rabbit and rat primary

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,
this kit is for mouse or rabbit primaries only. When we  do a single
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.
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu

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