Vitreoretinal VEGF expression and albumin leakage were determined by immunoblot analysis. On P17, the animals were killed by CO2 exposure, and retinal and vitreous tissue was isolated and homogenized by sonication at 4°C in lysis buffer (5 mM HEPES [pH 7.5], 50 mM NaCl, 0.5% Triton X-100, 0.25% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) containing 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM benzamidine, phosphatase inhibitor cocktails 1 and 2 (10 μL/mL; Sigma-Aldrich), and proteinase inhibitor cocktail set III (10 μL/mL; Calbiochem, San Diego, CA). The insoluble pellet was removed by centrifugation at 4°C, and the protein concentration of the supernatant was measured using a protein assay reagent kit (Bio-Rad Laboratories, Hercules, CA). Soluble protein (30 μg) was resolved by SDS-PAGE on 10% 1.0-mm 10-well minigels (NuPAGE Novex Bis-Tris; Invitrogen, Carlsbad, CA) and electrotransferred to a 0.2-μm-pore PVDF membrane (Invitrogen). The membrane was blotted with 1:1000 polyclonal goat anti-albumin antibody (Bethyl Laboratories, Montgomery, TX), 1:1000 monoclonal mouse anti-β-actin antibody (MA1–744; Affinity Bioreagents, CO), and 1:500 polyclonal rabbit anti-VEGF antibody (A2; Santa Cruz Biotechnologies). Peroxidase-linked anti-goat, -mouse, and -rabbit IgG antibodies (Amersham Biosciences, Buckinghamshire, UK) were used as secondary antibodies. Immunoreactive bands were detected by chemiluminescence (Super Signal West Dura Extended-Duration Substrate; Pierce, Rockford, IL). Images were captured (ChemiGenius Imaging Station; SynGene, Frederick, MD), and relative band density was determined (Genetools program; SynGene). The intensity of the β-actin signal was used as an endogenous control for loading. Data are expressed as the albumin:β-actin or VEGF:β-actin densitometric unit ratio.