I need some advice on the following experiment. We have fixed cells on coverslips that we need to examine with three specific antibodies. One is rabbit, two are mouse.
I can stain the rabbit with anti-rabbit alexa 488, and I can stain the first mouse antibody with anti-mouse alexa 594. After washing, I also want to have a third mouse antibody, but this antibody (to gamma-tubulin) will be conjugated directly to Alexa 647.
Can I do this? Obviously I am worried about the anti-mouse 594 going to the tubulin antibody, or that some of the tubulin might bind to the mouse 594 at that location. I haven't done 3 (4 with dapi) colour microscopy before....