Various eye tissues dissected from donor eyes were placed in lysis buffer (50 mM Tris, pH 8.0, 0.5% sodium dodecyl sulfate, 0.5% Triton X-100, 137 mM NaCl, 3 mM KCl, 8 mM Na
2HPO
4-7H
2O, 1 mM KH
2PO
4, protease inhibitors [Roche, Indianapolis, IN])
41 and homogenized using a rotor stator homogenizer (Polytron; Kinematica, Lucerne, Switzerland). Tissue lysates (15 μg) or AH volume containing 15 μg total protein were mixed in equal proportions with 2× Laemmli buffer, boiled, and separated on 4% to 15% SDS-PAGE gradient gels (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA) in 1× transfer buffer (50 mM Tris, 384 mM glycine, 0.01% SDS, 20% methanol). Membranes were blocked in 20 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween-20, and 2% nonfat evaporated milk. Blots were probed with rabbit polyclonal anti–human OPN (Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti–human GAPDH (Novus Biologicals, Littleton, CO), or mouse monoclonal anti–human serum albumin (Abcam, Cambridge, MA) antibodies, followed by a secondary horseradish peroxidase-linked anti–rabbit or anti–mouse antibody (GE Healthcare, Piscataway, NJ). Antibody/antigen complexes were detected using ECL Western blot signal detection reagent (GE Healthcare). Kodak film was used to visualize the protein signals (BioMax XAR; Eastman Kodak, Rochester, NY). Each film was digitized with a photographic scanner (Perfection 2400; Epson, Long Beach, CA). The band intensities in Western blot analysis were normalized to GAPDH (cell lysates) or human serum albumin (in AH) and quantified using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at
http://rsb.info.nih.gov/ij/index.html).