CUPROLINIC BLUE (QUINOLINIC PHTHALOCYANINE) COUNTERSTAINING OF ENTERIC NEURONS FOR PEROXIDASE IMMUNOCYTOCHEMISTRY

This report details a method for the use of Cuprolinic Blue (quinolini c phthalocyanine) as a counterstain for immunoperoxidase Phaseolus vul garis (PHA-L)-labeled fibers in gastrointestinal wholemounts. Cuprolin ic Blue (in the presence of high MgCl2 concentration) forms a particul arly stable blue chromophore with single-stranded RNA in neuronal Niss l substance and nucleoli. In contrast to the non-specific staining pro vided by traditional neuronal dyes, Cuprolinic Blue produces prominent enteric neuron staining with minimal coloration of the muscle and con nective tissue surrounding the ENS plexuses. However, this specific st aining pattern is not obtained when the dye is used as a neuronal coun terstain for immunoperoxidase-labeled material unless it is applied be fore the DAB chromagen. The counterstaining of gut wholemounts is opti mized by using 0.5% Cuprolinic Blue at 37 degrees C. A lower concentra tion of the dye and temperature is useful for the concomitant delineat ion of the PHA-L immunoperoxidase-labeled injection sites in brainstem sections. The protocols outlined-for Cuprolinic Blue counterstaining of both enteric and central nervous system material are also appropria te for the localization of other immunocytochemically identified antig ens.