For immunoperoxidase staining, retinal sections from normal and glaucomatouseyes were deparaffinized, rehydrated, and pretreated with 3% hydrogen peroxidein methanol to decrease endogenous peroxidase activity. Following washingwith phosphate-buffered saline solution containing 0.1% bovine serum albumin,the sections were incubated with 20% inactivated normal donkey serum (ChemiconInternational Inc, Temecula, Calif) for 30 minutes at room temperature toblock background staining. The sections were then incubated with a mouse monoclonalantibody to HIF-1α (1:1000; Novus Biologicals Inc, Littleton, Colo)for 16 hours at 4°C. After washing, the sections were incubated with thebiotinylated anti-mouse IgG (1:400; Chemicon International Inc) for 1 hourat room temperature and then with avidin-biotin complex (ABC reagent, VectastainABC Elite kit; Vector Laboratories, Burlingame, Calif) for 1 hour at roomtemperature. Following several washes, color was developed by incubation with3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St Louis, Mo) as cosubstratefor 5 to 7 minutes. Sections were counterstained with Mayer hematoxylin andmounted with Permount (Fisher Scientific, Pittsburgh, Pa). The primary antibodywas eliminated from the incubation medium, or mouse serum (Sigma-Aldrich)was used to replace the primary antibody to serve as the negative control.Slides were examined in a microscope (Nikon, Tokyo, Japan), and images wererecorded by digital photomicrography (Optronics, Goleta, Calif).