Retinal Flat Mount Immunostaining

Seven days after the intravitreal injections, mice (n = 6 mice/eyes per treatment group) were euthanized, their eyes were enucleated and immediately fixed overnight in 4% paraformaldehyde at 4°C. After three washes with 1x PBS, retinas were carefully separated from the globe and incubated overnight in blocking buffer (5% normal donkey serum and 5% BSA in PBS) at 4°C, cuts were made in the four quadrants (superior, inferior, nasal and temporal) and retinal flat mounts were prepared. The retinal flat mounts were then incubated with the primary antibody, goat anti-Brn3a (1:200, SC-31984, Santa Cruz Biotechnology, Inc.) for three days at 4°C. After three washes with PBS, the flat mounts were incubated overnight in the corresponding secondary antibodies: Alexa 488 conjugated donkey antigoat antibody (1:1000 dilution, A11055, Invitrogen) at 4°C. After washes, the flat mounts were mounted using Prolong Gold anti-fade (Life Technologies) and images were taken using the Keyence fluorescence microscope (Keyence, Osaka, Japan).