P iκb α b 9 sc 8404

BV-2 cells were seeded at a density of 5 × 10³ cells per well on a Millicell EZ slide (Millipore, Billerica, MA, USA) and grown to 80% confluence in LCM. After 12 h of serum starvation in FBS-free DMEM, cells were treated with Zinc20267861, Zinc18204217, and Zinc33254827 and incubated for 12 h. Following incubation, BV-2 cells were stimulated with 1 μg/ml LPS (Sigma) for 24 h. Samples were fixed with 2% formaldehyde (Sigma-Aldrich) for 10 min, permeabilized with 0.2% Triton-X100 (Sigma) in PBS for 15 min, and then blocked with 5% NGS (Thermo Fisher Scientific) for 1 h at RT. The samples were 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 overnight; then, they were visualized DyLight 647 and 488, 1:1000 (Thermo Fisher Scientific). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) 1:5000 (Sigma-Aldrich) and mounted ProLong Diamond antifade reagent (Invitrogen, Thermo Fisher Scientific).

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).