Sz stb1

The eyeballs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 12 h. Under an Olympus SZ-STB1 (Olympus) dissection microscope, the anterior segment tissues, vitreous, and retinas were removed to isolate the RCSC. Approximately four to eight relaxing radial incisions were made, and the remaining RCSC were incubated overnight in a blocking solution composed of 5% NGS (Thermo Fisher Scientific) with 0.01% Triton-X (Sigma-Aldrich). The RPE-choroidal-scleral complexes were then incubated with beta-amyloid (36-6900; 1:100; Thermo Fisher Scientific), crystallin alpha B (Ab151722, 1:50; Abcam), Iba1 (019-19741; 1:100; Wako), and Anti-CD4 (MA5-12259, 1:100, Thermo Fisher Scientific). The RPE-choroidal-scleral complexes were then incubated with primary antibody dissolved in 2.5% NGS (Thermo Fisher Scientific) for 24 h before being washed three times for 10 min with PBS-T; then, the samples were incubated for 24 h with Cy5 conjugated secondary antirabbit (ab97077) and antimouse (ab6563) secondary antibody 1:1,000 in 2.5% NGS (Thermo Fisher Scientific) and DAPI 1:5,000 (Sigma-Aldrich). After another PBS-T washing, three times for 10 min, the samples were mounted on slides with ProLong Diamond antifade reagent (Invitrogen).

Mice were euthanized by CO2 inhalation. Eyeballs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 12 h. Under an Olympus SZ-STB1 (Olympus) dissection microscope, the anterior segment tissues, vitreous, and retinas were removed to isolate the retinal pigment epithelia choroid-scleral complex (RCSC). Approximately four to eight relaxing radial incisions were created, and the remaining RCSC were incubated overnight in a blocking solution composed of 5% NGS (Thermo Fisher Scientific) with 0.01% Triton-X (Sigma-Aldrich). The RPE–choroidal-scleral complexes were then incubated with COX-2 (MA5-14568; 1:1000; Thermo Fisher Scientific) and p-IκB-α (B-9) (sc-8404; 1:100; Santa Cruz Biotechnology, CA, USA) antibody for 24 h; following washing three times for 10 min with PBS-T, the samples were incubated for 24 h with DyLight 647 and 488, 1:1000; (Thermo Fisher Scientific) and DAPI 1:5000 (Sigma-Aldrich). Following another wash with PBS-T, the whole mounts were counterstained with DAPI and mounted with ProLong Diamond antifade reagent (Thermo Fisher Scientific).