Eye sections from hyperoxia-exposed neonatal mice at different time points were deparaffinized in solvent and clearing agent (Citrisolv; Fisher Scientific) and were rehydrated in graded ethanols. After epitope retrieval with citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6, 30 minutes, 95°C–100°C), sections were blocked with goat serum and incubated with rabbit anti-AQP1 (1:500; Chemicon, Temecula, CA) and isolectin B4 (Sigma). Sections of extraocular tissues (skeletal muscle and kidney) from embryonic day (E) 16.5 mice were processed identically and incubated with rabbit anti-AQP1 and rat anti-mouse CD31/PECAM (PharMingen, San Diego, CA). Primary antibodies were detected with Texas Red goat anti-rabbit and Alexa-488 goat anti-rat secondary antibodies, and isolectin B4 was detected with Alexa-488 streptavidin (all at 1:200; Molecular Probes). For immunostaining of retinal endothelial cell cultures, cells were fixed for 15 minutes with paraformaldehyde at room temperature, blocked with bovine serum, and incubated with rabbit anti-AQP1, rat anti-mouse CD31/PECAM, or rabbit anti-human von Willebrand factor (VWF; 1:50; Dako, Carpinteria, CA) and then incubated with secondary antibodies as described. Colocalization studies of AQP1 and VWF were performed using labeling reagents (Zenon Technology; Molecular Probes). Antibody specificity was confirmed by excluding primary or secondary antibodies.