Quantification of Laser-Induced Choroidal Neovascularization in Mice

Mouse eyes were harvested on day 14 post L-CNV induction. The eyes were enucleated and fixed in 4% PFA in PBS for 1 h at 4°C. The anterior portion including lens and the retina were removed, then the posterior eyecups were dissected out and underwent further fixation in 4% PFA in PBS overnight. The fixed eye cups were washed in blocking buffer (0.3% Triton X-100, 5% bovine serum albumin (BSA) in PBS) for 2 h at 4°C. The eye cups were then stained for vasculature using the rhodamine-labeled Ricinus communis agglutinin I (Vector Labs, Burlingame, CA, USA) and Alexa Fluor 488 conjugated-Isolectin B4 from Griffonia simplicifolia (GS-IB4) (Molecular Probes, Thermo Fisher Scientific) at 1:250 dilution in buffer containing 0.3% Triton X-100, 0.5% BSA in PBS, overnight at 4°C. The posterior eyecups were washed three times with PBS and mounted in fluorescent mounting medium (VectaShield; Vector Laboratories, Inc.) and cover-slipped. Confocal imaging and analysis of L-CNV lesion volume were performed as previously described [53 (link)]. Treatments were compared by unpaired t-test (two tailed) with Welch’s correction, while mouse body weights were compared by two-way repeated-measures ANOVA with Holm-Sidak post hoc tests using GraphPad Prism v. 6.