On days 1, 7, and 14 after laser photocoagulation, the eyes were enucleated and the sensory retina and retinal pigment epithelium (RPE)–choroid complex were carefully isolated, placed in 150 μL of lysis buffer (20 mM imidazole hydrochloric acid (HCl), 10 mM potassium chloride (KCl), 1 mM magnesium chloride (MgCl2), 10 mM ethyleneglycol-bis [beta-aminoethylether]-N, N'-tetraacetic acid, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM ethylenediaminetetraacetic acid with protease inhibitor; Sigma-Aldrich, Tokyo, Japan) and sonicated on ice for 15 seconds. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and the VEGF levels in the supernatant were determined with a rabbit VEGF ELISA kit (detection range 1.56–100 pg/mL; Cusabio Biotech Co. Ltd., Tokyo, Japan) at 450 to 570 nm and an absorption spectrophotometer (SpectraMax 34000A ROM, version 2.04; Bio-Rad, Hercules, CA, USA) and normalized to total protein according to the manufacturer's protocol. A standard curve was plotted from the measurements of diluted standard solutions (7.8–500 pg/mL), and the concentration of VEGF in each sample was determined in comparison with this curve.