Mice were euthanized under isoflurane, and eyeballs enucleated. For histopathological analysis, eyeballs were post-fixed in Davidson's solution overnight followed by 10% neutral buffered formalin overnight, and then embedded in paraffin. The sections were stained with hematoxylin and eosin. Immunohistochemistry for complement component 3 (C3) was performed using anti-C3 antibody (EPR19394; Abcam, Cambridge, UK) with the VENTANA automated slide stainers (Ventana Medical Systems Inc, Tucson, AZ, USA). Infiltrating cells and C3 deposition were morphologically evaluated by pathologists (S.O. and K.Y.), and retinal thickness was measured using ImageJ (NIH, Bethesda, MD, USA). For glial fibrillary acidic protein (GFAP) staining, eyeballs were post-fixed with phosphate-buffered saline (PBS)-diluted 4% paraformaldehyde (15710; Electron Microscopy Sciences, Hatfield, PA, USA) for 2 hours on ice. After washing in PBS, retinas were flat mounted and permeabilized with blocking buffer (10% fetal bovine serum [F2442; Merck, Darmstadt, Germany] and 0.2% Triton X-100 [X100; Merck] in PBS) for 1 hour at room temperature. Retinas were then incubated with anti-GFAP monoclonal antibody (1:250, 13-0300; Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight.
26 After washing with PBS, the retinas were incubated with secondary antibody (1:250, A48262; Thermo Fisher Scientific) and Hoechst (1:1000, 346-07951; Dojindo Laboratories, Kumamoto, Japan) at 4°C overnight. PBS-washed samples were then mounted with ProLongGlass Antifade Mountant (P36980; Thermo Fisher Scientific) and observed under a confocal microscope (A1R; Nikon, Tokyo, Japan).