Choroidal Flat Mount Preparation and Imaging

Choroidal flat mount preparation, staining, and imaging were undertaken as previously described [21 (link)]. Briefly, eyes were enucleated at 7 day after laser injury and immediately fixed for 1 h in a solution of 4% paraformaldehyde in phosphate-buffered saline (PBS; 9 g/L NaCl, 0.232 g/L KH₂PO4, and 0.703 g/L Na²HPO4; pH 7.3). The anterior segment and crystalline lens were removed, and the retinas were detached and separated from the optic nerve head with a pair of fine-curved scissors. The remaining eye cups were washed with cold ICC buffer (0.5% BSA, 0.2% Tween 20, and 0.05% sodium azide) in PBS. Next, a 1 : 500 dilution of isolectin B4 (Sigma-Aldrich, USA) and 1 : 1000 dilution of CD31 (Abcam, Cambridge, UK) were incubated at 4°C overnight and then washed with cold PBS buffer. A 1 : 1000 dilution of fluorescence-conjugated secondary antibody (Abcam, Cambridge, UK) was incubated for 1 hour and then washed with cold PBS buffer. Radial cuts were made toward the optic nerve head, and the sclera-choroidal/RPE complexes were flat mounted, covered, and sealed. Image J software was used to analyze fluorescence images. The summation of the whole stained area in each section multiplied by the distance between sections (1 μm) was used as an index for the CNV lesion volume. The volumes of the all lesions in each eye were averaged and considered as an n = 1 for statistical analysis.