P2ry12

All antibodies used at 1:200: CD11b (Clone M1/70, Biolegend, Cat 10137), CD45 (Clone 30‐F11, eBioscience, Cat 56–0451–82), MHCII (Clone M5/114.15.2, Biolegend, Cat 107626), CD80 (Clone 16‐10A1, Biolegend, Cat 104706), CD103 (Clone 2E7, eBioscience, Cat 48‐1031‐80), CD8a (Clone 53‐6.7, Biolegend, Cat 100712), CD4 (Clone RM4‐5, BD Biosciences, Cat 550954), CD44 (Clone IM7, Biolegend Cat 103011), P2RY12 (Clone S16007D, Biolegend Cat 848006), IFNγ (Clone XMG1.2, Biolegend Cat 505826), CD68 (Clone FA‐11, Biolegend Cat 137017). WNV‐specific CD8+ T cells were identified with fluorescent‐labeled immunodominant Db‐restricted NS4B peptide.

After isolating on the first day, a small part of mixed retinal cells was seeded at a density of 1 × 10⁶ cells per well in a 24-well plate with poly-L-lysine-coated glass coverslips at the bottom. For cultured retinal microglia, we cultured them after shaking on day 17 at a density of 2 × 10⁵ cells per well onto poly-L-lysine-coated glass coverslips in a 24-well plate. After attachment to the coverslips, cells were deprived of medium and fixed with 4% PFA for 1 h. Then, 0.1% Triton was used for permeabilization for 20 min. Then, 5% bovine serum albumin (BSA, Sigma) was performed as a blocking buffer. Primary antibodies of anti-ionized calcium-binding adaptor molecule 1 (Iba1, Abcam, #ab178846, 1:200) and anti-Purinergic Receptor P2Y12 (P2RY12, Biolegend, #848001, 1:100) were used to stain the microglia-related marker at 4°C overnight. Primary antibodies were incubated at 4°C overnight. The next day, coverslips or tissue sections were incubated with secondary antibodies (Alexa Fluor 488 of goat anti-mouse, Yeasen, 1:1,000; Alexa Fluor 647 of goat anti-rabbit, Yeasen, 1:1,000) at room temperature for 1 h. DAPI (Abcam, #ab228549, 1:1,000) was used for staining nuclear DNA. The morphology of retinal microglia and their specific locations in retina layers were observed under immunofluorescence microscopy (Leica) equipped with the NIS-Elements Advanced Research software.