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You didn't say whether your primary antibodies are made in the same species
or if they have the same isotype. If they are made in different species say
one is mouse the other rat then life will be easier, you could buy one
second step reagent against rat and one against mouse making sure that the
anti-rat reagent is absorbed against mouse Igs and vice versa. In this
scenario you can add both primarys together AND both secondaries together.
If your primary antibodies are made in the same species but have different
isotypes then you can do the same thing buying isotype specific secondary
antibodies (absorbed against the other isotype).
If they are of the same species and isotype then you have problems, if you
could conjugate one of your antibodies to a fluorochrome or biotin it can
be done, that would be the way to go probably. If you do conjugate one AB
and the primary antibodies are mouse then you can use the following method:
1. Prepare cells in the usual way, spin them down and aspirate supernatant.
2. Incubate cells first with the unconjugated antibody, wash then incubate
with the second step reagent (usually goat anti mouse-fitc or PE) and wash
again (twice).
3. Incubate cells with 20=B5l 10% mouse serum (heat inactivated) in PBS for
10 minutes at room temperature.
4. Add without washing your directly conjugated antibody, incubate as
usual, wash and fix.
Good luck
Simon
Simon Monard
=46ACS Lab Manager
Aaron Diamond Center for AIDS Research
455 First Avenue
New York
NY 10016
Ph (212) 448 5141
=46X (212) 725 1126
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