Yes, you can couple both primary antibodies directly with different fluorophores:

E.g. Antibody X with Alexa Fluor 488, and antibody y with Alexa Fluor 594 for example.

Then you can perform the IF directly: Then you do not need labeled secondary antibodies against rabbit IgG which would recognize both antibodies at the same time, but the signal comes directly from the labeled primary antibody

hi there

 the zenon technology is quiet a useful way to do this. i have attached a link.  i hope this answers your question.

http://www.lifetechnologies.com/uk/en/home/references/molecular-probes-the-handbook/antibodies-avidins-lectins-and-related-products/zenon-technology-versatile-reagents-for-immunolabeling.html

Have you checked to see if either of the two proteins of interest are made from another host from another company?  I agree with Khamal about the zenon technology.  I have used it and it works.  However, the staining with the zenon is not as strong as without.  Therefore, if you are going to use zenon, choose the antibody that is more abundantly expressed of the 2 antibodies you are studying.  If one of your antibodies is a cellular marker, that would be perfect. If you have any questions about using the zenon, do not hesitate to ask.  Good luck!

thank you all for the suggestions

All above a re great. A most simple way I have done this, but it doesn't always work, but most often it has worked for me: I do one staining first - primary and secondary then wash then block and then do the second staining.

Additionally if you have monoclonal antibodies and one is for example IgG2a and the other one is IgG1 or 2b you can have very specific secondary antibodies that will not cross react with the subtypes of IgG.

Hope that's helpful.

I support Kalinas answer. If both of your primarys have the same isotype (and - as Kalina mentioned - not only the same host, which would enable the use of isotpe-specific secondaries (but probably not suitable for rabbit)) you can stain the cells with your first antibody, wash, stain with your first fluorochrome-conjugated anti-rabbit antibody at the optimal (titrated) concentration, wash, then block free rabbit antibodies on the cell surface with an excess of unconjugated anti-rabbit antibody, wash extensively, stain with your second rabbit antibody, wash and finally stain with your second fluorochrome-conjugated anti-rabbit antibody. (May accordingly be modified for intracellular staining.)

Personally, I'd confide in such a protocol for qualitative analyses, however, I think it is not optimal for quantitative analyses (i.e. statements on the amount of the respective antigens of interest).

Pre-labelling of both individual primary antibodies with different fluorochromes - like Marc-David has mentioned - would IMHO be the better alternative if you go for quantitative analyses.

Best regards!

I recommand FAB-secondary ab. You remember the Y-structure of antibodies. FABs have only one binding-arm. That prevents from co-binding of the second primary-ab.Start detecting your first primary-ab with FAB-secondary and then detect the second primary-ab with  an anti-rabbit IgG. Jackson Laboratories (Dianova in Germany) offer a lot of fluochrome and biotine  labelled FABs. If your signal is not so intensive you can do an enhancement with Streptavidin-Flu or - POX via biotinylated FABs.

In the attachment you will find the principle of this methode and the references.

Good Luck!