intraperitoneal injection of 25 mg Etanercept and were eutha- nized 24 hours later, perfused with PBS, and retinas were isolated under a dissecting microscope. Control mice (n = 2) received an equal volume of normal saline (N/S). Retinas from each group were pooled in 0.5 ml chilled lysis buffer and homogenized with a tissue grinder. Supernatants were collected after centrifugation at 15,000 rpm for 10 min at 4uC. Protein concentrations were determined by Bio-Rad protein assay (Hercules, CA). Equal amounts (500 mg) of the retinal lysates were incubated overnight at 4uC with 20 ml of protein A/G agarose beads (Protein A/G Plus; Pierce, Thermo Scientific). The beads were then washed seven times with chilled lysis buffer and immunopellets were separated by SDS-PAGE and transferred onto PVDF membranes, where they were probed overnight at 4uC with a mouse anti-human IgG1 Fc region antibody (R&D Systems). Membranes were then incubated with an HRP-conjugated donkey anti-mouse secondary antibody (Jackson Immunoresearch), developed with ECL, and exposed to Fuji RX autoradiographic film. ELISA Analysis for TNF-a The tissue complex containing the vitreous and neural retina was collected at 3, 7, 14, 21, or 28 days after EVC. Proteins were extracted from each retina individually in 200 ml PBS containing a protease inhibitor mixture (Complete, Roche Diagnostics, Plea- santon, CA) and sonicated (10 W, 5 s, 4uC; Sonifier 250, Branson, Danbury, CT). The supernatant was collected after centrifugation at 14,0006g for 20 min at 4uC (Micromax RF, IEC, Needham Heights, MA), and the total protein concentration was measured with the DC protein assay kit (Bio-Rad, Hercules, CA). The level of TNF-a was determined with rat TNF-a enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer’s protocols with slight modification. TNF-a References 1. 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