Detection of melanin-mediated false-positive for BaxΔ2 immunohistochemical staining in human skin tissue

Bax{Delta}2 is a pro-apoptotic isoform of the Bax family. We have previously shown that Bax{Delta}2 protein expression is low in most healthy human tissues with the protein mainly scattered in the connective tissues. Surprisingly, in skin tissue, the Bax{Delta}2-positive staining was strikingly strong, especially in the basal layer of the epidermis. It has been documented that melanin in the basal cells displays a brown color that could give false-positive staining in the commonly used DAB-based (3-3-diaminobenzidine) immunostaining. To avoid the false-positive staining from DAB-melanin, we performed immunofluorescent staining on the same set of tissues which were positive for Bax{Delta}2 in the DAB immunostaining. The results from co-immunofluorescent staining of Bax{Delta}2 and a basal cell marker CK-14 showed that the previously detected DAB-stained Bax{Delta}2-positive epidermal cells were CK-14 positive but Bax{Delta}2 negative. Consistent with our previous finding, the Bax{Delta}2-positive cells in the dermis connective tissues are positive in both DAB-based and fluorescence-based immunostainings. These data indicate that the DAB-stained Bax{Delta}2 positive cells in the basal layer of skin tissue are false-positive due to a misinterpretation of the melanin color, but the dermal connective tissue still presents real Bax{Delta}2 positive staining.