Confocal microscope - (Dec/08/2006 )

Hi!

I'm trying to carry out some confocal microscope studies. I'd like to analyze colocation of 2 proteins using a secondary antibody linked to FITC and other secondary antibody linked to rhodamin. The problem is I've realised that my primary antibodies, both are from rabbit. So my question is; can I use 2 primary antibodies from the same specy (rabbit) in confocal colocation studies? Is there some possibility to use them?
Thank you very much!!

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I don't think it will work, because the secondary antibodies recognize both primary ones. You'll never get a specific "colour" for the proteins then, they will both be labeled with the FITC and Rhodamin.

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It might work if they have different isotypes and you can use isotype-specific secondaries. Otherwise it could be possible to biotinylate one of the antiodies and use a streptavidin-secondary or directly conjugate with a flurochrome using a kit. Otherise it might be easier to get an antibody from a different species.

Ceri

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Thank you!!!

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try to label primary antibodies directly, either with Alex dye (do not use FITC or rhodamin), there is a kit from Molecular Probes/Invitrogen, or

try to pre-incubate your primary antibodies with each its secondary antibody, and apply these complexes to your fixed cells/tissues

or find a mouse monoclonal to substitute one of your rabbit polyclonal

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