Mmp 9

We performed Western blot analysis as previously described [17 (link)]. Briefly, the supernatant samples of the retina (equal amounts of protein samples) were loaded onto a BIS-TRIS Blot Gel (Life Technologies, Carlsbad, CA, USA) and electrophoresed. Following separation, the proteins were transferred to a nitrocellulose membrane and incubated with 5% skimmed milk at 4°C overnight. After blocking, the membranes were incubated at 25°C for 1 h with primary antibodies against iNOS (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), matrix metalloproteinase-2 (MMP-2) (1:1,000; Bioss Inc., Woburn, MA, USA), MMP-9 (1:1,000; Abnova Co., Taipei, Taiwan), vascular endothelial growth factor (VEGF) (1:1,000; Abnova Co.), macrophages (F4/80, 1:1,000; Bio-Rad, Raleigh, NC, USA), β-amyloid (1:1,000; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), and β-actin (1:5,000; Sigma-Aldrich, St. Louis, MO, USA). Bands on the membranes were visualized using a horseradish peroxidase-conjugated secondary antibody (Life Technologies, Frederick, MD, USA) and ImmunoStar Zeta reagent (Wako, Osaka, Japan). Images were acquired using the Multi-Gauge Software program (Fujifilm, Greenwood, SC, USA).