Simultaneous immunofluorescent labeling using anti-BrdU monoclonal antibody and a melanocyte-specific marker in formalin-fixed paraffin-embedded human skin samples.

Abstract

Immunolabeling of tissue sections requires careful optimization of protocols in order to achieve accurate and consistent data. Multiple immunolabeling is desirable when determining the exact location and phenotype of cell populations in the same cellular compartment. 5-bromodeoxyuridine (BrdU)-immunolabeling is commonly used to assess cellular proliferation in vitro. However, the technical limitations of standard methods preclude multiple antigen immunolabeling. The aim was therefore to develop a robust protocol for simultaneous labeling using anti-BrdU and a melanocyte-specific marker in formalin-fixed paraffin-embedded (FFPE) skin samples. Human skin samples were obtained from patients undergoing elective plastic surgery. The tissue was incubated with BrdU, and a standard sample procedure for FFPE tissue was used. Heat-induced antigen retrieval was performed in a conventional pressure cooker, followed by immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies. Fluorescent-conjugated secondary antibodies were used for signal detection. We have demonstrated both proliferating cells (BrdU-immunopositive) and melanocytes (Melan A/MART-1-immunopositive) in the basal compartment of the epidermis in our skin samples. Successful double labeling requires heat-induced epitope retrieval to replace the harsh pretreatment protocols of standard BrdU immunolabeling methods. We have optimized a robust protocol for the double labeling of proliferating cells and cells bearing melanocyte-specific antigens (melanocytes and/or melanoblasts) in FFPE human skin samples.