At P12, mice with oxygen-induced ischemic retinopathy began receiving daily intraperitoneal (systemic) injections of 0, 0.1, 1, or 5 mg/kg of acriflavine or they received a single intravitreous (intraocular) injection of 0, 1, 10, or 50 ng of acriflavine. At P17, mice were euthanized, retinas were dissected and stained with FITC-labeled Griffonia Simplicifolia lectin, and flat mounted. With our staining protocol, there is selective staining of retinal NV and hyaloid vessels but no staining of pre-existent retinal vessels. (A) Systemically administered acriflavine suppressed retinal NV: the retina from a mouse treated with 5mg/kg/day of acriflavine (left) shows little retinal NV compared with a retina from a mouse treated with PBS injections (middle) and compared with controls, the mean (±SEM) area of NV per retina was significantly less than control in retinas from mice treated with 0.1, 1 or 5 mg/kg (*p<0.05 for comparison with control, **p<0.01 for comparison with control). (B) Intraocular injection of acriflavine suppressed retinal NV: the retina from an eye injected with 50ng of acriflavine (left) shows little NV compared with the retina from an eye injected with PBS (middle) and the mean (±SEM) area of NV per retina was significantly less than that in control retinas for eyes injected with 10 or 50 ng, but not 1 ng (right, *p<0.01 by ANOVA with Dunnett’s correction). (C) Systemically administered acriflavine suppressed choroidal NV: the mean (±SEM) area of choroidal NV was significantly less (right, p=0.018 by Wilcoxon matched-pairs signed-ranks test) in eyes from mice treated with 5 mg/kg acriflavine (left) compared with eyes from control PBS-injected mice (middle). (D) Intraocular injection of acriflavine suppressed choroidal NV: the mean (±SEM) area of choroidal NV was significantly less (right, p=0.01 for difference from control by unpaired t-test) in eyes injected with 50 ng of acriflavine (left) compared with those injected with PBS (middle).