Dm550b fluorescence microscope

Mice were killed by CO2 asphyxiation and eyes were enucleated. Eyes were fixed for 1 h in 4% PFA, then rinsed and sectioned at the limbus; the cornea and lens were discarded. Eyecups were incubated in 30% sucrose overnight at 4 °C, then embedded in OCT and sectioned (10 µm). Cryosections were blocked with PBS containing 1% horse serum, 0.1% Triton 1 h at room temperature and exposed overnight to rabbit anti-IAB1 antibody (019-19741, 1:200, Fujifilm Wako) at 4 °C. After washing, sections were incubated 2 h with an Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500, ThermoFisher Scientific) and counterstained with Hoechst 33342 (1:1000, ThermoFisher Scientific). The slides were then washed, mounted, and viewed and photographed with a Leica DM550B fluorescence microscope (Leica Biosystems).

Mice were killed by CO2 asphyxiation and eyes were enucleated. The globes were fixed in 4% PFA for 45 min, and then sectioned at the limbus; the cornea and lens were discarded. RPE/choroid tissues were separated from retina and incubated overnight with rabbit anti-IBA1 antibody (019-19741, 1:400, Fujifilm Wako) and Alexa Fluor 594 phalloidin (1:100, ThermoFisher Scientific) in PBS containing 0.1% Triton. Tissues were rinsed and incubated 2 h with an Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500, ThermoFisher Scientific) and counterstained with Hoechst 33342 (1:1000, ThermoFisher Scientific). RPE/choroids were flatmounted, viewed and photographed with a Leica DM550B fluorescence microscope (Leica Biosystems). MPs were counted on whole RPE/choroidal flatmounts. RPE nucleation and morphology were evaluated on randomized photos taken between optic nerve and the mid-periphery retina. Melanophages on RPE/choroidal flatmounts were defined and quantified as IBA1⁺ MPs (green fluorescence) that visibly block the red Alexa Fluor 594-phalloidin fluorescence of the underlying RPE when viewed in the red channel.