C57BL/6 mice were obtained from Central Lab. Animal. OIR was induced in newborn mice as previously described [16 (link),17 (link)]. At postnatal day (P)14, we intravitreally injected 1 μL of phosphate-buffered saline (PBS) or anti-mouse VEGF164 antibody (1 μg/eye; cat. no.: AF-493-NA, R&D) into the right eye of mice using NanoFil 10 μL syringe with 35 gauge needle (WPI) after anesthesia using tiletamine plus zolazepam (Zoletil 50, Virbac; 30 mg/Kg) and xylazine (Rompun, Bayer; 10 mg/Kg). At P17, the mice were euthanized by carbon dioxide in deep anesthesia using tiletamine plus zolazepam and xylazine. Then, the enucleated eyes were prepared for immunofluorescent staining of whole mount retinas with Alexa Fluor 594 isolectin GS-IB4 conjugate (5 μg/mL; Invitrogen). The whole mount retinas were viewed with a fluorescence microscope (Eclipse 90i; Nikon). Then, the neovascular tufts were marked and the extent of them were calculated using NIS-Elements AR (v. 3.2; Nikon). The area of neovascular tufts was normalized to the area of whole retina. All animal studies were approved by the Seoul National University Institutional Animal Care and Use Committee and conducted in agreement with the ARVO statement for the use of animals in ophthalmic and vision research.
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