To measure NgR1 in the retina, the animals were euthanized at 0 and 4 weeks after laser coagulation and treatments with PBS or sNgR-Fc. The retinas were dissected and homogenized in lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1mM EGTA) supplemented with 10% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail from Sigma (St. Louis, MO). After centrifugation at 13,000 rpm for 30 minutes to remove cell debris, the protein concentration of the supernatant was measured using a protein assay kit (Bio-Rad DC; Bio-Rad Laboratories, Hercules, CA). A 40 to 80 μg aliquot of proteins from each sample was subjected to 6% (for NgR1) or 10% (for β-actin) SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membrane. The membranes were blocked with blocking buffer (Odyssey; LI-COR, Inc., Lincoln, NE) for 1 hour at room temperature. Incubation with mouse anti-NgR1 (1:100; Biogen Idec, Inc.) antibody or mouse antibody against actin (1:2000; Chemicon, Hofheim, Germany) was performed for 16 hours at 4°C. After washing, the membranes were incubated with goat anti-mouse secondary antibody 1:2000 (IRDye 800CW; LI-COR, Inc.) in blocking buffer (Odyssey; LI-COR, Inc.) for 1 hour at room temperature. After washing, the membranes were scanned and analyzed using an infrared imaging system (Odyssey Infrared Imaging System; LI-COR, Inc.). All experiments for Western blot analysis were performed with 5 animals in each group and repeated 2 to 3 times.