PSMA expression in the primary prostatic lesion: immunohistochemistry and immunofluorescent staining

Tissue samples were fixed with 5% formaldehyde, embedded in paraffin, and cut into consecutive 5 μm sections using standard procedures. The sections were incubated first with primary anti-PSMA monoclonal antibody overnight at 4°C. Then after three washes with PBS, sections were incubated with horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature. The bound antibody complexes were detected using a diaminobenzidine detection system, after which the slides were counterstained with hematoxylin. Alternatively, for immunofluorescent staining, sections were incubated with a fluorescently labeled secondary antibody for 1h at room temperature in the dark, after which nuclei were counterstained using 4, 6-diamidino-2-phenylindole. Immunostained images were observed with microscope (Leica DM IL).