Paraffin-embedded tissue sections (5 μm) were deparaffinized using xylene and alcohol and incubated with a blocking solution containing 3% bovine serum albumin (BSA; Sigma-Aldrich Corp.) in PBS for 30 minutes. The primary microglial cultures and cocultures of the cortical explants and microglia were fixed in 4% paraformaldehyde in 0.1 M PBS for 15 minutes, then washed with PBS and incubated with a blocking solution containing 1% BSA and 0.1% Triton X-100 (Sigma-Aldrich Corp.) in PBS at 37°C for 1 hour. After samples were blocked, they were incubated overnight at 4°C with the following primary antibodies: anti-ionized calcium binding adaptor molecule 1 (Iba1; Wako, Osaka, Japan), anti-CD68 (Bio-Rad, Hercules, CA, USA), anti-9F5 (Japan patent number P4815610), anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich Corp.), anti-microtubule-associated protein 2 (MAP2; Sigma-Aldrich Corp.), and anti-β-tubulin III (Tuj1; Sigma-Aldrich Corp.). After samples were washed with PBS, they were incubated for 1 hour at room temperature with the following secondary antibodies: fluorescent dye Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 594-conjugated anti-rabbit IgG (Thermo Fisher, Rockford, IL, USA). Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Fluorescent images were captured using a BX51 (Olympus, Tokyo, Japan) or BZ-X710 (Keyence, Osaka, Japan) fluorescence microscope. Images of the optic nerve region were taken 500 μm distal to the choroidal plane.