Anti arg1 antibody

For cryosections, eyes were removed carefully and embedded in optimal cutting temperature compound (Sakura Fine Technical). The frozen samples were then sliced transversely (8 μm) with a cryostat (Leica CM1950) at –20 °C. Only the cross-sections throughout optic nerve were used for staining and analysis. For retinal-RPE flat-mount, the eyes were fixed in 4% PFA for 60 min at room temperature and the RPE-sclera complex were dissected out. The cryosection, RPE flat-mount sheet samples and ARPE cells were blocked with 0.5% Triton-X100/5% BSA and incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-ZO-1 antibody (Santa Cruz, sc-33725), anti-Arg1 antibody (Santa Cruz, sc-18355), anti-Nos2 antibody (Santa Cruz, sc-7271), anti-IL-4Ra antibody (Santa Cruz, sc-28361), anti-IL-4 antibody (Santa Cruz, sc-53084), and anti-p-Nrf2 antibody (Abcam, ab76026). Then they were incubated with secondary antibodies for 1 h and counterstained with DAPI (Invitrogen, D1306) for 5 min before mounted. Negative control was done by omission of the primary antibody. The images were obtained using a confocal microscope (LSM880, Carl Zeiss).

The immunofluorescence analysis was carried out according to previously published methodology [50 (link)]. Anti-68 antibody (Santa Cruz Biotechnology) and anti-iNOS antibody (Santa Cruz Biotechnology) and anti-Arg-1 antibody (Santa Cruz Biotechnology) were incubated in a humidified chamber at 37 °C O/N on the sections. After washing with PBS, sections were incubated for 1 h at 37 °C with secondary antibodies, TEXAS RED-conjugated anti-rabbit Alexa Fluor-594 (Molecular Probes, Eugene, OR, USA) and FITC-conjugated anti-mouse Alexa Fluor-488 (Molecular Probes, Eugene, OR, USA). Nuclei were stained by adding 2 μg/mL 40,60-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) in PBS. Slides were then washed with PBS and incubated with a secondary antibody. Specific labeling was identified with an avidin–biotin-peroxidase complex and a biotin-conjugated goat anti-rabbit immunoglobulin G (Vector Lab, Milan, Italy) [51 (link)]. Stained sections were observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy). Each specimen was observed in five random visual fields and the average number of double-positive cells in each specimen was calculated [19 (link)].

Recombinant human CX3CL1 (cat#300-31) was from Peprotech; IL-4 (cat#12340045) was from Immunotools; LPS (cat#L4391) was from Sigma-Aldrich; anti-Arg1 antibody was from Santa Cruz (cat#sc-271430 RRID:AB_10648473); anti-Actin antibody (cat#A2066) was from Sigma-Aldrich. Secondary antibodies were from DAKO; Microbeads CD11b+ were from Miltenyi Biotec. All cell culture media, fetal bovine serum (FBS), goat serum, penicillin G, streptomycin, glutamine and Hoechst (cat#33342, RRID:AB_10626776) were from Invitrogen; poly-L-lysine (cat#P2636) and papain were from Sigma-Aldrich. Griess reagent kit for Nitrite determination was from Molecular Probe (cat#G-7921), Lactate Assay Kit (cat#MAK064) and Arginase Activity Assay Kit (cat#MAK112) were from Sigma-Aldrich; Seahorse Cell Mito Stress Test Kit for Seahorse XFe Analyzer Respiratory Assay reagents were from Agilent (cat#103015-100).