Confirmation of specific immunoreactivity of PPZ0506 antibody against human, mouse, and rat ERβ proteins in immunocytochemical analyses. (a) Immunocytochemical detection of human, mouse, and rat ERβ proteins in transfected COS-7 cells using anti-human ERβ monoclonal antibody (PPZ0506). (b) Immunocytochemical detection of FLAG-tagged ERα and ERβ proteins in transfected COS-7 cells using anti-DYKDDDDK monoclonal antibody (2H8). Transfected cells were treated with 10 nM E2 (+) or 0.1% EtOH (–). “h”, “m”, and “r” indicate human, mouse, and rat, respectively. Mock-transfected cells (mock) were used as negative controls. Alexa Fluor 488 and 4′,6-diamino-2-phenylindole (DAPI) images were pseudocolored in green and red, respectively. Scale bar: 50 μm. Similar results were obtained in three separate experiments (n = 3).