To assess expression of multiple proteins in prostate tumors, we developed a multiplex, multiround IF protocol to visualize expression of eight markers simultaneously: nuclear marker 4′,6‐diamidino‐2‐phenylindole (DAPI), nucleolar marker nucleolin, epithelial marker PanCK, and prostate‐specific markers prostate‐specific antigen (PSA), prostate‐specific membrane antigen (PSMA), androgen receptor (AR), prostein, and alpha‐methylacyl‐CoA racemase (AMACR). The gene discussed is AR; the disease is prostate neoplasm.