Anti mouse il 1β

For surface staining, cryostat sections (10μm thick) of lymph nodes were dried, fixed in 4% PFA for 10 minutes, blocked with PBS 5% BSA and then incubated with the following anti-mouse antibodies in 1% BSA for 1 hour: B220 Pacific Blue (RA3-6 B2, Biolegend), CD169 AF488 (Ser-4, ATCC), IgD FITC (11-26c.2a, BD Biosciences), Langerin AF647 (929F3.01, Dendritics) and GL-7 AF647 (GL7, Biolegend). For intracellular staining, sections were permeabilized with PBS Triton 0.3% for 5 minutes before blocking and then incubated with anti-mouse IL-1β (polyclonal, R&D), IL-18 (polyclonal, Abcam) or IL-33 (polyclonal, R&D), followed by incubation with AF555 anti-goat or anti-rabbit IgG (Invitrogen). CD1d tetramer staining was performed as previously described (Lee et al., 2015 (link)). Briefly, lymph nodes were incubated overnight with PE-labeled PBS-57 loaded CD1d tetramer in 2% FCS at 4°C. Next day, lymph nodes were washed and fixed with 4% PFA for 1 hour and snap frozen. 10μm sections were blocked with 5% BSA and stained with anti-PE antibody (polyclonal, Novus Biologicals) followed by goat anti-rabbit AF555 (Life Technology). Imaging was carried out on a LSM 780 (Zeiss) inverted confocal microscope using a Plan-Apochromat 40x NA 1.3 oil immersion objective.

For human specimens, the IHC detection was conducted using antibodies anti-human HIF-1α (Abcam, Cambridge, MA, USA), anti-human NLRP3 (LifeSpan BioSciences, Seattle, WA, USA), anti-human cleaved-caspase-1 (Biorbyt Ltd., Cambridge, UK) and anti-human IL-1β (Abcam; mainly detect mature IL-1β). Images were captured by Nikon Eclipse 80i (Nikon Instruments Inc., Melville, NY, USA) and shown in Figure 1 and Supplementary Figure 1. For mouse tissues, the IHC was detected using anti-mouse NLRP3 (LifeSpan BioSciences, Seattle, WA, USA), anti-mouse IL-1β (R&D Systems Inc., Minneapolis, MN, USA; detect pro- and mature IL-1β). Images were captured by Aperio® AT2 (Leica Biosystems, Wetzlar, Germany), and shown in Figure 4 and Supplementary Figure 2. The IHC scores were determined as described previously (Kreisberg et al., 2004 (link)).

ATP, nigericin, and DMSO were obtained from Sigma. SiO₂, Pam3CSK4, poly (dA:dT), and ultrapure lipopolysaccharide (LPS) were provided by InvivoGen. Dihydrotanshinone I (DHT) was purchased from MCE and Targetmol. Salmonella was supplied as a gift from Dr. Tao Li from the National Center of Biomedical Analysis (Beijing, China). MitoSOX was supplied by Invitrogen. Anti-mouse caspase-1 (1:1,000, AG-20B-0042) and anti-NLRP3 (1:2000, AG-20B-0014) were supplied from AdipoGen. Anti-ASC (1:1,000, #67824) and anti-GAPDH (1:1,000, #5174) were supplied by Cell Signaling Technology. Anti-mouse IL-1β (1:1,000, AF-401-SP) was obtained from R&D Systems. Anti-flag (1:1,000, 80010-1-RR), anti-NEK7 (1:1,000), and anti-lamin B (1:1,000, 10895-1-AP) were supplied by Proteintech. F4/80 (565,410) was obtained from BD Biosciences.