A simple method for immunocytochemical staining with multiple rabbit polyclonal antibodies.

We report a single step, simple, repeatable, rapid and reliable technique for simultaneous immunocytochemical staining with two or more rabbit polyclonal antibodies. This technique, which we have dubbed the "Pretty Poly" method, is based on conjugating the antibodies with commercially available, fluorophore-tagged Staphylococcal protein-A (SP-A). Staining is illustrated at the calyx type presynaptic nerve terminal of the chick ciliary ganglion with antibodies directed against three nerve terminal proteins: neurofilaments of the axonal cytoskeleton, and two secretory vesicle proteins, SV2 and cysteine string protein (CSP). Images were deblurred with an iterative deconvolution protocol. Staining with a single polyclonal antibody was bright and had a resolution approaching light microscope limit. Treatment with two different polyclonal antibodies conjugated with contrasting dye-tagged protein-A resulted in double staining without significant crossover that was fully equivalent to the standard primary/secondary technique. The same single step protocol was used to stain with all three rabbit polyclonal antibodies or to combine the technique with a standard monoclonal primary/secondary antibody stain. Thus, the Pretty Poly protocol is a highly flexible, simple and yet effective staining technique that essentially solves the problem of co-staining with multiple polyclonal rabbit antibodies.