Immunohistochemistry was performed on the tissue sections of TMAs of the patients with PCa to gauge the expression levels of NRCAM. Following deparaffinization and dehydration, sections were processed to reveal antigens using a microwave oven. Briefly, the sections were blocked and then incubated with primary antibody against NRCAM (1:100, cat. no. ab87427; Abcam). The slides were subsequently incubated with anti-rabbit secondary antibody and visualized by DakoCytomation Liquid DAB plus Substrate Chromogen System (DakoCytomation).
Two pathologists, who were blind to patient data independently evaluated the TMA samples. The immunohistochemistry score was calculated by the sum of the staining intensity and the fraction of positive tumor cells as previously described (11 (link)–13 (link)).