Preparation of retinal pigment epithelium (RPE)/Choroid flat mounts and Quantification of CNV volume

As described previously⁷, eyes were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 1 hours. The cornea, lens, the vitreous and the retina were then removed, and the posterior eyecups containing the RPE/choroid/sclera were fixed in 4% paraformaldehyde for additional 1 hour. After fixation, the posterior eyecups were blocked in PBS containing 1% bovine serum albumin (BSA) and 0.5% TritonX-100 for 30 mins at room temperature. To label invading choroidal vessels, the eyecups were then incubated with AlexaFluor 568-conjugated Isolectin B4 (1:200, Invitrogen, Carlsbad, CA) overnight at 4 °C. An anti-GFP antibody (1:500, ABCAM, Cambridge, MA) was co-incubated with isolectin B4 to label GFP in RPE. After staining, the eyecup was flattened by cutting radial incisions and flatmounted onto a microscope slide with vectashield mounting medium (Vector Laboratories, Burlingame, CA) for confocal imaging. Confocal z-stack images of each lesion in two channels (568 nm, 488 nm) were acquired using a 20X objective (Olympus Fluoview, Center Valley, PA). Images were imported into IMARIS and lesion volume was measured using the “surfaces” module (Version 9.1.2, Bitplane, Santa Barbara, California, USA). An additional surface was created for the GFP channel (488 nm) to give a rough quantification of VMD2 activation in the immediate area of the lesion. Lesions with obvious hemorrhage or bridging CNV were not included for analysis.