Retinal Microglia Quantification in Retinal Angiomatous Proliferation

For fluorescence microscopy, the mice were perfused with PBS followed by 4% paraformaldehyde (PFA). After enucleation, eyes were fixed in 4% PFA for 1 h at 4 °C in the dark and retinal flatmounts were dissected prior to staining as previously described [34 (link),35 (link)]. Primary antibodies against IBA1 (AB178846, Abcam, Cambridge, United Kingdom) and Collagen IV (COL4, AB769, Merck Millipore, Billerica, MA, USA) were added over two nights at a dilution of 1:500 at 4 °C in the dark. Secondary antibodies were added at a dilution of 1:500 (Alexa Fluor^® 647 and Alexa Flour^® 488, Life Technologies, Carlsbad, CA, USA) overnight at 4 °C in the dark. Retinal flatmounts were imaged using a Hamamatsu NanoZoomer S60 (Hamamatsu Photonics, Herrsching, Germany). To quantify microglia cell numbers, confocal images of RAP lesions were taken using a Leica SP8 confocal microscope with a 20× objective lens (Leica, Wetzlar, Germany). RAP lesions were detected when focusing below the outer plexiforme layer and were defined as neovascularizations originating from retinal vessels and extending into the normal avascular outer nuclear layer and reaching the retinal pigmented epithelium.