*5.12. Immunohistochemical Analysis*

Deparaffinized tissue arrays were incubated with anti-LSD1 (1:200; Cell Signaling), anti-Nkx3.1 (1:400; Cell Signaling), or anti-synaptophysin (1:100; Cell Signaling). Deparaffinized animal tissues were incubated with anti-CD31 (1:100; Santa Cruz). Antibody binding was visualized by a conventional immunostaining method, as described previously [48,49], using an autoimmunostaining apparatus (HX System, Ventana, Tucson, AZ, USA).

LSD1 expression was evaluated using intensity scores for normal prostate glands and carcinoma cores from patients. For LSD1 immunoreactivity in nuclei, raw nuclear intensity data for tumor cells in prostate cancer cores and luminal cells in normal prostate glands were measured using a BZ-9000 multifunctional microscope and analysis software (Keyence Japan, Osaka, Japan). For each patient, evaluations were repeated five times and an average intensity score was calculated for each core.
