Re: incubating conjugated with UNconjugated antibody!

Raquel,

A few different approaches all of which we have used:

A. Indirect staining
1. Perform intracellular staining with unlabeled mAb
2. wash
3. Stain with secondary Goat anti-mouse IgG PE
4. wash
5. Incubate with 2% mouse serum
6. Incubate with your directly labeled surface mAb

B. Zenon
http://probes.invitrogen.com/products/zenon/

C. Can also use subclass specific secondary Ab if the two primary mAbs are a
different subclass (i.e. IgG1 vs. IgG2a). Then you don't need to do the
serum block IF it is really subclass specific.

Generally the indirect staining has better S/N than the zenon, but that may
not be an issue if your target is brightly expressed. PE conjugates are
generally best. Needs lots of controls as specificity is a major issue. If
you have any recombinant source of the intracellular molecule, best to show
you can block staining by preincubating your mAb with a large (100-1000 fold
molar excess) of the recombinant molecule.

For details read methods:
Foster, B., L. B. Schwartz, G. Devouassoux, D. D. Metcalfe, and C. Prussin.
2002. Characterization of mast-cell tryptase-expressing peripheral blood
cells as basophils. J Allergy Clin Immunol 109:287-293.

_______________________
> Calman Prussin 
> Laboratory of Allergic Diseases
> National Institute of Allergy and Infectious Diseases/ NIH

> From: Raquel Maia <hemato_hc@inca.gov.br>
> Date: 21-Nov-2006 23:10:25 ZW3
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: incubating conjugated with UNconjugated antibody!
> 
> 
> Hi Flowers
> I'm desperate because i need to do a double staining using one conjugated
> antibody for surface and another UNconjugated (because there is no
> conjugated antibody)antibody for intracellular staining and then add a
> secondary antibody! what will happen? Is it tottaly wrong?
> Please help me!
> 
> Flavia C Vasconcelos