Azure B as a counterstain in the immunohistological evaluation of heavily pigmented nevomelanocytic lesions

Heavily pigmented melanocytes and melanophages are often difficult to distinguish from one another. Several methods have been used to address this problem, including bleaching the tissue sections and immunohistologic stains. The stains are plagued, however, by a resemblance of the precipitates of common chromogens—such as diaminobenzidine (DAB)—to melanin with hematoxylin counterstains. It has been reported previously that azure B allows the distinction between melanin and chromogenic precipitates by metachromatically staining melanin granules. In this study, 35 heavily pigmented lesions, including 27 melanomas. 6 blue nevi, and 2 pigmented spindle-cell nevi, were labeled with an antibody to S-100 protein and with HMB-45; they were then counterstained in parallel with Harris’ hematoxylin and with azure B. Comparison showed that precipitates of DAB could be easily separated from metachromatically labeled melanin in all azure B–stained cases, whereas that distinction remained difficult when hematoxylin was used as a counterstain. These results confirm the effectiveness of azure B as an aid in interpreting immunohistologic preparations of melanocytic lesions.