Use of the peroxidase-antiperoxidase technique for differential staining of multiple cell types in the rat pancreatic islet.

Multiple staining of the endocrine cells of the pancreatic islet was studied in tissue obtained from adult rats. After fixation in Bouin's fluid and processing for light microscopy, the unlabeled peroxidase-antiperoxidase (PAP) technique was performed. Successful staining procedures used variations of the PAP technique with 3,3'-diaminobenzidine (DAB) and 4-chloro-1-naphthol (CN) as chromagens. The purpose of this study was to stain as many of the four primary cell types (A-cells, B-cells, D-cells, PP-cells) as possible either simultaneously or sequentially using photomicroscopy. At optimum antibody titer, there was minimal nonspecific background staining which made it possible to differentiate cell types by intensity of the chromagen. Any two cell types can be shown by using DAB with the first antibody and CN with the second. To demonstrate three cell types simultaneously, three methods which altered dilutions and chromagens were used. The first method consisted of decreasing dilutions of primary antibody with DAB and CN as the chromagens. The second method involved repetitive DAB applications resulting in three intensities of brown. The third method used a DAB immersion after the second cell type was stained. This produced a color differential so the third cell type could be distinguished with CN. To demonstrate the three cell types sequentially, a masking technique was introduced with photomicroscopy. In order to block the preceding complex, the previous cell type (demonstrated by CN) was restained with DAB at an increased dilution. The next cell type was then stained with CN. These four methods were tried in attempts to stain four cell types in the same tissue section.