Goat anti il33

Paraffin-embedded lung sections (4-µm thickness) were used for this study. Images were taken under polarized light using an upright dry 40× objective. Tissues were deparaffinized and after antigen retrieval, permeabilized with 0.4% triton X-100. Tissues were blocked with 10% BSA and maintained in PBS + 5% BSA + 0.4% triton X-100 throughout antibody treatments. Primary antibodies were incubated at 4°C overnight and secondary antibodies were incubated for 1 hr at room temperature. Primary antibodies used (1:200 dilution) include rabbit anti-NFκB p65 (#8242, Cells Signaling Technology); mouse anti-cRel (#MA5-15859, Thermo Fisher Scientific); rabbit anti-NFκB1 (#13586, Cells Signaling Technology); mouse anti-NFκB1 (#NBP2-66976, Novus); rabbit anti-RUNX1(#ab229482, Abcam); goat anti-IL33 (#AF3626 R&D); mouse anti-ICAM1 (#sc-1511, Santa Cruz Biotechnology, Inc); and mouse anti-CD3 ((# sc-7296, Santa Cruz Biotechnology, Inc). Goat anti-rabbit and anti-mouse IgG-A594 or IgG-A488 were used as secondary antibodies (1:200 dilution at RT). For anti-goat, rabbit anti-goat IgGA-647 was used as secondary antibody (1:200 dilution at RT). DAPI was used for nuclear counterstaining. The ProLong Gold antifade reagent was used as mounting media. Mount slides were examined with a Leica DM6000 B. Slide Book 6 (3i) was used for analysis and capturing images.

Brain sections were blocked with 5% donkey serum in PBS for 1 hour, followed by overnight incubation (4°C) with the primary antibodies. After washing, sections were incubated for 1 hour at 20°C with secondary antibodies conjugated with fluorophores (1:1000, Jackson ImmunoResearch Laboratories, Inc.). Fluorescence images were captured with an Olympus Fluoview FV1000 confocal microscope and FV10-ASW 2.0 software (Olympus America). Primary antibodies used in this study include: Goat anti-IL-33 (R&D System), mouse anti-APC (MilliporeSigma), rabbit anti-GFAP (Dako), mouse anti-NeuN (MilliporeSigma), and rabbit anti-Iba1 (Wako).

Wild-type (WT), CMO and CMO/MC^– mice were monitored for the initial appearance of a tail kink or paw deformity to record the date of disease onset. For tissue cytokine measurements, equal weights of distal tail segments and hind paws were snap-frozen, homogenized and assayed for IL-1β (e-bioscience) using ELISA. To distinguish levels of pro- and mature forms of IL-1β and IL-33, some homogenates were analyzed by immunoblot using mouse anti-IL-1β (3A6, Cell Signaling Technology) and goat anti-IL-33 (R&D Systems); anti-β-actin (Santa Cruz Biotechnology) served as a loading control.