Eyes that underwent the ischemia–reperfusion injury were enucleated at 3, 6, 12, 24, and 48 hours after reperfusion, and eyes from siRNA-treated rats were enucleated at 12 and 24 hours after reperfusion. Retinas from normal rats were also studied. The sensory retina was immediately removed after enucleation and homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, and 1 mM EDTA [pH 7.4]) containing protease inhibitors (1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) and a phosphatase inhibitor (1 mM Na3VO4, and 1 mM NaF). The supernatant was collected after centrifugation at 15,000 rpm for 15 minutes. Equal amounts of protein (75 μg) were loaded onto a 12% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. After incubation with 3% nonfat dried milk in 0.05% Tween 20 and Tris-buffered saline (TBST), the membranes were incubated with 1:500 monoclonal mouse anti-HO-1 antibody, or 1:2000 polyclonal rabbit anti-HO-2 antibody (Stressgen, Victoria, British Columbia, Canada), or 1:3000 monoclonal mouse anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO) with 1% bovine serum albumin (BSA) in TBST at 4°C for 12 hours. After a wash in TBST, the membranes were incubated with horseradish-conjugated secondary antibodies at a dilution of 1:4000 for 1 hour. The chemiluminescence Western blot analysis system (ECL; Amersham Biosciences, Buckinghamshire, UK) was used for signal detection.