Immunohistochemistry of Mouse Posterior Eyecup

Freshly dissected whole eyes were fixed in 4% paraformaldehyde (PFA) (J61899-AP, Alfa Aesar) for 2 hours and then the anterior parts were removed. The posterior eyecups were infiltrated with 5%, 10%, and 20% sucrose in PBS for 30 minutes each at room temperature, and the samples were finally infiltrated with 2:1 mixture of 20% sucrose in PBS and OCT compound (Sakura Finetek) for 45 minutes and frozen completely (1–3 minutes) on dry ice. Immunofluorescence was performed on frozen sections from the posterior eyecups as described previously (70 (link)). The sections were incubated with PBS containing 5% normal donkey serum for 30 minutes and then incubated overnight at 4°C with primary antibodies for STING (NBP2-24683, Novus Biologicals) and CD31 (11-0311-82, Thermo Fisher Scientific) diluted 1:200. The sections were washed with PBS and then incubated with 1 μg/mL DAPI (62248, Thermo Fisher Scientific) and goat anti-rabbit Alexa Fluor 555–conjugated secondary antibody (1:1000) (A21428, Thermo Fisher Scientific). Images were acquired by a Zeiss LSM 710 confocal workstation.