Cuprolinic Blue (quinolinic phthalocyanine) counterstaining of enteric neurons for peroxidase immunocytochemistry

This report details a method for the use of Cuprolinic Blue (quinolinic phthalocyanine) as a counterstain for immunoperoxidase Phaseolus vulgaris (PHA-L)-labeled fibers in gastrointestinal wholemounts. Cuprolinic Blue (in the presence of high MgCl2 concentration) forms a particularly stable blue chromophore with single-stranded RNA in neuronal Nissl substance and nucleoli. In contrast to the non-specific staining provided by traditional neuronal dyes, Cuprolinic Blue produces prominent enteric neuron staining with minimal coloration of the muscle and connective tissue surrounding the ENS plexuses. However, this specific staining pattern is not obtained when the dye is used as a neuronal counterstain for immunoperoxidase-labeled material unless it is applied before the DAB chromagen. The counterstaining of gut wholemounts is optimized by using 0.5% Cuprolinic Blue at 37 degrees C. A lower concentration of the dye and temperature is useful for the concomitant delineation 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 appropriate for the localization of other immunocytochemically identified antigens.