Steve,

You may not want to try this approach given the small amounts of
material you have, but it is very easy to biotinylate one of your
primaries (assuming at least some purification) using a succinimidyl
ester (Molecular Probes or Pierce). After labeling with the
non-biotin primary and secondary, you can then use your
biotin-primary with streptavidin-fluor or its cousins.

In the same context, you could directly fluor label each primary with
a different color and do the labeling in one step. Again to be
practical, the antibodies need to be moderately purified. Using
succinimidyl esters and small salt exchange columns (Pierce has some
ready made) for removal of excess fluors, you can prepare the
conjugated antibodies in a couple of hours. It's really not hard.

Caveats:   1) if you get it wrong, you can kill the primary
        2) if the primary isn't purified it may be hard to get the fluor on
        because of background competition
        3) if you've only got 20 ug of Ab, then you get one shot.

Potential Pluses: One blocking step, one primary incubation, one wash
sequence, probably the lowest non-specific background

I think that people should consider labeling their primaries more
often. It may not be appropriate for you, but I think in many cases
like yours it can simplify multiple labeling protocols with only
modest effort.

>I am doing some labeling of tissues with two rabbit derived antibodies,
>one against serotonin and one against a peptide called Tpep. I need to do
>a double labeling experiment in order to determine whether both these
>antigens reside in the same or different neurons and their axons.
>
>Two questions
>
>1. Can anyone suggest a reliable method for double labeling using a
>secondary fluor-conjugated probe with two different antibodies derived
>from the same species (rabbit)? I don't have a large supply of either of
>these antibodies.
>
>
>Steve Kempf
>
>_________________________________________________________________________
>The mind is like a parachute; it works much better when it's open.
>~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>
>Stephen C. Kempf                             Tel: 334-844-3924
>Associate Professor                          Fax: 334-844-4065
>Depart. of Zoology and Wildlife Science
>101 Cary Hall
>Auburn University, AL  36849
>USA                                        Email: [log in to unmask]
>Director - AU Hybridoma Facility - www.auburn.edu/research/hybridoma/
>
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Mario M. Moronne, Ph.D.
Material and Life Science Div.  M/S 6-2100
University of California
Berkeley Lab
1 Cyclotron Rd.
Berkeley, CA
94720

ph (510) 486-4236
FAX (510) 528-8076
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