Immunofluorescence staining was performed as reported previously [9 (link)]. In brief, rat CMECs on coverslips were washed once with PBS, fixed in 4% paraformaldehyde (pH 7.2~7.6) for 20 min at room temperature, washed with PBS, and then blocked with 5% bovine serum albumin in TBST for 15 min. The cells were incubated with PBS (negative control) or mouse antibody against estrogen receptor alpha (1: 100, Santa Cruz, Santacruz, CA, US) overnight at 4°C and then for 1 h at 37°C, then washed with PBS 3 times for 5 min each. The cells were then incubated with FITC-labeled goat anti-mouse IgG (1: 200 in TBST, Santa Cruz, Santacruz, CA, US) for 45 min and washed 3 times. Coverslips were analyzed using DPController and DPManager software (Olympus, Tokyo, Japan).