| | Re: Multiple antigen immunostaining
Well, in my opinion, fluorescence is the way to go. Because the fluorfores on each antigen of interest will fluoresce at different wavelengths, fluorescent or confocal microscopes will be able to image each antigen staining individually, and if properly done, take the guesswork out of discriminating between your two signals.
If you don't have access to fluorescent scopes, then two color IHC is an alternative (as you suggest).
I would first optimize each antigen individually, and be sure to choose color that are easily distinguishable from each other. for example...don't choose brown and red. Next to each other, they both look brick colored.
However, you may find you need to "retrieve" the antigen if they've been fixed in paraffin. If you google antigen rescue, you'll find many common methods. In our hands heating the slides in sodium citrate buffer works well. Keep in mind the optimal antigen retrieval time may be different for each of your antigens (if it's required at all), in which case you'll have to choose a happy medium between the two times.
Give it a try and let us know how it goes.