Anti il 33

Western blot analysis was performed to assess protein levels of apoptosis and autophagy-related proteins in the brains of RNS and control, using our standard methods as described previously (Gao et al., 2017 (link)). In brief, proteins were extracted from brain tissue and equal amounts of protein were separated by gel electrophoresis and transferred onto Hybond-polyvinylidenedifluoride (PVDF) membranes. After incubating with primary antibodies to anti-IL-33 (1:500, R&D), anti-NF-κB (1:500, CST), anti-ST2L (1:500, abcam), anti-LC3B (1:3000, abcam), anti-Beclin-1 (1:1000, bioworld), anti-P62 (1:1000, abcam), anti-cleaved-caspase-3 (1:500, bioworld), anti-Bcl-2 (1:500, abcam) and anti-β-actin (1:10,000, Sigma). anti-β-actin was used as a loading control. Then, the PVDFs were incubated with the respective HRP-conjugated secondary antibody for 2 h at room temperature. Blots were detected with the ECL chemiluminescence system (Beyotime Institute of Biotechnology) and were captured on autoradio graphic films (Kodak). Films were scanned and densitometric analysis of the bands was performed with Sigma Scan Pro 5.

For detection of IL‐23 and IL‐23R expression, MLE‐12 cells (SV40‐transformed mouse‐derived alveolar epithelial cell line; American Type Culture Collection) were grown in Dulbecco's modified Eagle medium:Ham's F‐12 with 2% fetalbovine serum in a humidified atmosphere at 37°C with 5% CO2. The cells were then stimulated with low‐dose Dp (10 μg/ml) or PM (0.1 μg/ml) for overnight. The expression of IL‐23 was detected in cytosol using NE‐PER Extraction Reagent (Thermo Fisher Scientific) and IL‐23 ELISA kit (Biolegend). For expression of IL‐23R, cells were stained with PE‐conjugated anti‐IL‐23R (Thermo Fisher Scientific). For IL‐23 siRNA experiment, MLE12 cells were treated with IL‐23p19 siRNA (Santa Cruz, 30 nM/well) or normal control (NC, Santa Cruz) siRNA using transfectamine (Invitrogen) and after 5 hours, the cells were washed and stimulated low‐dose Dp (10 μg/ml) or PM (0.1 μg/ml) for overnight. The protein level of IL‐23, GMCSF in cytosol and IL‐33 in the nucleus was measured using NE‐PER Extraction Reagent (Thermo) and ELISA kit (R&D). For detection of intracellular IL‐33 expression from MLE‐12 cells, the cells were permeabilized and incubated with anti‐IL‐33 (1:100, R&D), 647‐conjugated anti‐goat IgG (1:500, Invitrogen). Each sample was read on LSRII (BD Biosciences).

Antibodies were purchased from BD Pharmingen unless mentioned otherwise. RPE cells were harvested and washed before staining with anti-IL-33 (R&D System) or ST2 (MD Biosciences) antibodies. To examine the leukocyte phenotype in the DLN and spleen, single-cell suspensions were stained with anti-F4/80, anti-CD206 (MR, Serotec) and anti-CD273 (PD-L2, eBioscience) antibodies. For measurement of intracellular cytokines, cells were restimulated with 50 ng/mL phorbol-12-myristate-13-acetate and 500 ng/mL ionomycin (both from Sigma) in the presence of Golgi-Stop (BD Bioscience) for 4 h. Cells were then harvested and stained with cell surface anti-CD4 antibody before being permeabilized with Perm/Fix solution (eBioscience) and finally stained with anti-IL-17A (clone eBio17B7), anti-IFN-γ (clone XMG1.2), anti-IL-5 (TRFK4), and anti-IL-4 (clone 11B11) antibodies (eBioscience). Isotype-matched IgG antibodies from eBioscience were used as negative controls. Cells were analyzed by FACS Calibur using CellQuest software (BD Biosciences).

Immunofluorescent analysis was performed to identify IL-33 and ST2L IR cell type in brain by standard methods, as described previously (Gao et al., 2017 (link)). Briefly, brains were perfused pericardially with PBS followed by 4% paraformaldehyde and cut into 10-μm sections with a cryotome. The sections were incubated with primary antibody with anti-IL-33 (1:100; R&D), anti-ST2L (1:200; Abcam), anti-NeuN (1:200; Abcam), anti-GFAP (1:500; Abcam), anti-Iba-1 (1:50; Abcam), then followed by a mixture of fluorescein isothiocyanate and tetra methyl rhodamine isothiocyanate conjugated-conjugated secondary antibodies for 2 h at 4°C, respectively.