Af1460

Primary antibodies: rat anti-CD31 (DM3614P, Dianova, Hamburg, Germany), goat anti-CD32b (AF1460, R&D Systems, Minneapolis, MN, USA), goat anti-Lama4 (AF3837, R&D Systems, Minneapolis, MN, USA), rabbit anti-Desmin (ab32362, Abcam, Cambridge, UK), rabbit anti-CD3 (100202, Biolegend, San Diego, CA, USA), rat anti-CD4 (100402, Biolegend, San Diego, CA, USA), rat anti-CD8a (100802, Biolegend, San Diego, CA, USA), rabbit anti-F4/80 (30325S, Cell Signaling, Danvers, MA, USA), rat anti-CD11b (101202, Biolegend, San Diego, CA, USA), rabbit anti-CD11c (97585S, Cell Signaling, Danvers, MA, USA), rat anti-MHCII (14-5321-85, eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-FoxP3 (12653, Cell Signaling, Danvers, MA, USA), rabbit anti-CD45 (ab10558, abcam, Cambridge, UK), rat anti-Ly6C (ab15627, abcam, Cambridge, UK), rat anti-Gr1 (ab 25377, abcam, Cambridge, UK), goat anti-Reelin (AF3820, R&D Systems, Minneapolis, MN, USA), goat anti-Periostin (AF2955, R&D Systems, Minneapolis, MN, USA), rabbit anti-TGFBI (ab170874, abcam, Cambridge, UK). Secondary antibodies: donkey Alexa-Fluor 488, Alexa-Fluor 647, and Cy3-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany).

Western blot (WB) analysis was performed on protein extracted from post-ONC mouse retinas, primary microglia, and RGCs as previously described [28 (link)]. Washed and blocked polyvinylidene fluoride membranes from WBs were incubated with primary antibodies. Primary antibodies included anti-Sema3A (1:1000, rabbit monoclonal, Abcam, ab23393), anti-CD16/32 (1:1000, goat polyclonal, R&D, AF1460), anti-CD206 (1:500, rabbit monoclonal, Abcam, ab125028), anti-Iba1 (1:1000, rabbit monoclonal, Abcam, ab178846), anti-SMI32(1:1000, rabbit monoclonal, Abcam, ab207176), and anti-Map2 (1:1000, rabbit monoclonal, CST, #4542). Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States) in TBS-T for 1.5 h at room temperature. ECL-Plus reagent (Bioground) was applied to the membranes and chemiluminescence visualized by a Fluor-S-max imager (Bio-Rad).

Primary antibodies included anti-Sema3A (1:100, rabbit monoclonal, Abcam, ab23393), anti-CD16/32 (1:100, goat polyclonal, R&D, AF1460), anti-CD206 (1:100, mouse monoclonal, Abcam, ab8918), anti-Iba1 (1:100, rabbit monoclonal, Abcam, ab178846), and anti-P2RY12 (5 µg/ml, mouse monoclonal, Biolegend, 848001). Secondary antibodies included anti-mouse IgG, anti-goat IgG, anti-Rat IgG, and Alexa Fluor 488, 594, and 647 conjugated to anti-rabbit IgGs (1:1000, Invitrogen). Mouse retinas were fixed in 4 % paraformaldehyde and cryo-sectioned at a thickness of 10 μm. Retina cryosections and fixed primary microglia were incubated with 0.1 % Triton X-100 (Sigma-Aldrich) in PBS at room temperature for 10 min, then incubated with primary antibodies at 4 °C overnight. Secondary antibodies were applied for 1 h at room temperature. The slides were mounted after nuclei counterstaining (Hoechst 1:2000). Images were taken by an SP-8 confocal microscope (Leica, Germany). For quantification of immunofluorescence, CD16/32⁺, CD206⁺, and Iba1⁺ cells were counted in 3 s. (10 μm thick) of retinal tissue per mouse. The mean number of cells per field was determined. All data were expressed as mean (n = 3 mice per group) ± SEM. The sections were selected randomly and were analyzed under a Leica SP8 confocal laser scanning microscope. Quantification of cell numbers was analyzed using ImageJ software.