Fluorescence of bisazo dye reaction products from the coupled tetrazonium method for proteins.

The coupled tetrazonium reaction is a well known histochemical method for proteins. This method, using Fast Blue B salt and 1-naphthol, has been applied on paraffin sections of grasshopper testis and rabbit trachea, as well as on horse blood smears. Microscopic observation under bright field illumination revealed the expected purple staining of protein-rich cell and tissue structures, which also showed a strong red fluorescence under ultraviolet, violet, violet-blue and green exciting light. Some weakly stained cell components (e.g., meiotic spindles) were easily visualized by fluorescence microscopy. Control preparations lacking either the tetrazonium or naphthol treatment, and spectroscopic studies on the bisazo dye produced in vitro (showing an emission peak at 660 nm) confirmed that the red fluorescence of stained structures arises from the protein-tetrazonium-naphthol reaction product formed in situ.