Anti mhcii

Methods for these experiments were adopted from our group’s previous work (9 (link)). Briefly, FLS obtained from RA patients were cultured on coverslips and treated with either RA NETs or vehicle. For plasma membrane detection, cells were incubated with membrane-dye (Biotium) for 30 min at 37 C. Cell were washed and fixed with 4% paraformaldehyde 12h at 4°C. For intracellular detection, cells were permeabilized with 0.2% triton for 10 min at room temperature. Coverslips were blocked with porcine gelatin (Sigma) for 30 minutes, then incubated for 1 hour with the primary antibody [anti-LL37 (Abcam) or anti-MHCII (Abcam)] at 37°C. After washing, coverslips were incubated for 30 minutes with secondary antibodies, then counterstained with Hoechst 1:1000. After further washing, coverslips were mounted on glass slides using Prolong-gold (Invitrogen). Images were acquired on a Zeiss LSM 780 confocal microscope.

Protein was isolated from human subcutaneous differentiated adipocytes (Zen-Bio) using RIPA lysis buffer, treated with recombinant IFNγ (R & D), IFNγ + JAK inhibitor (Sigma), or IFNγ + STAT1 siRNA (Dharmacon) and IFNγ + scrambled RNA and subjected to Western blotting performed as previously described^{51 (link)}, using primary antibodies from Cell Signaling Technology: anti-phospho-STAT1 (Y701) (1:1000, 9171), anti-STAT1 (1:1000, 9172), and anti- MHCII (1:3000, 68258), from Abcam: anti-HLA-DPB1 (1:5000, ab157210), and from Santa Cruz: anti-β-actin (1:5000, sc-47778).

Human postmortem brain tissue were supplied by the UK Multiple Sclerosis Tissue Bank (UK Multicentre Research Ethics Committee, MREC/02/2/39), funded by the Multiple Sclerosis Society of Great Britain and Northern Ireland (registered charity 207,495). Fresh-frozen blocks containing chronic active lesion from progressive MS patients were sectioned (10-μm), PFA-fixed, blocked in PBS with 10% normal horse serum (NHS) and immunostained with rabbit CHI3L1 (monoclonal antibody) 1:200 (Abcam) followed by biotinylated secondary antibody (Jackson Immunoresearch Laboratories, Cambridgeshire, UK), avidin/biotin staining (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine (DAB) staining (Vector Laboratories, Burlingame, CA). The RNAscope 2.5 Duplex Assay (ACD Biosystems) was performed according to the ACD protocol for fresh-frozen tissue. Chronic active lesions were hybridized with two mRNA probes per experiment. Hs-GFAP (Cat No. 311801) was used as the astrocyte marker together with Hs-CHI3L1 (Cat No. 408121). The probes were amplified according to manufacturer’s instructions and labeled with the following red or green color for each experiment. The Hs-CHI3L1 probe was also combined with immunohistochemistry (anti-GFAP and anti-MHCII, Abcam) as described above.