Goat anti il 1β antibody

Liver tissues were fixed in 4% (v/v) paraformaldehyde (PFA) in PBS and embedded with paraffin, which were then sliced into tissue sections (4 μM) and mounted on glass slides. These tissue slides were stained with goat anti-IL-1β antibody (1:100, R&D Systems) overnight at 4°C after a 20 min wash with 3% H₂O₂ and 30 min blocking with 10% serum and then probed with anti-goat Ig-G second antibody labeled with HRP according to the protocols described previously [32 (link), 40 (link)]. Negative controls were prepared without the primary antibodies. The area percentage of the positive staining were calculated in Image Pro Plus 6.0 software.

To confirmNLRP3 inflammasome formation in HSCs in mouse liver tissue, frozen tissue slides were stained with goat anti-IL-1β antibody (1:100, R&D Systems) and rabbit anti-desmin antibody (1:100, Thermo Fisher) overnight at 4°C after a 30 min blocking with 10% serum and then probed with Alexa 488- or Alexa-555-labeled secondary antibodies (1:500, Invitrogen). Similarly, cells were incubated with goat anti-NLRP3 antibody (1:200, Abcam), rabbit anti-ASC antibody (1:200, Santa Cruz), or rabbit anti-caspase-1 antibody (1:200, Santa Cruz) as we described previously [40 (link), 51 (link)]. Co-localization of IL-1β vs. desmin, NLRP3 vs. ASC or caspase-1 was calculated in Image Pro Plus 6.0 software, and the co-localization coefficient was calculated as the Pearson's correlation coefficient.

Liver tissues were fixed in 4% (v/v) paraformaldehyde (PFA) in PBS and embedded with paraffin, which were then sliced into tissue sections (4 μM) and mounted on glass slides. These tissue slides were stained with goat anti-IL-1β antibody (1 : 100, R&D Systems) overnight at 4°C after a 20 min wash with 3% H₂O₂ and 30 min blocking with 10% serum and then probed with anti-goat Ig-G second antibody labeled with HRP according to the protocols described previously [18 (link), 22 (link)]. Negative controls were prepared without the primary antibodies. The area percentage of the positive staining was calculated in Image Pro Plus 6.0 software.

Total protein was extracted from mouse lungs and cells by RIPA. Protein concentration was detected by the bicinchoninic acid assay (BCA, Thermo Fisher Scientific). Equal amounts of protein (30 µg) were loaded on SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Zhou et al. 2016 (link)). After blocking with skim milk (5%) in Tris-buffered saline (TBST) for 1 h at room temperature, the membrane was probed with the primary antibodies at 4 °C overnight: rabbit anti-NLRP3 antibody (1:2000; CST, Danvers, MA), rabbit anti-Nrf2 antibody (1:1000, CST), rabbit anti-HO-1 antibody (1:1000, ABCAm), goat anti‐IL‐1β antibody (1:2000; R&D, Minneapolis, MN), rabbit anti‐caspase‐1 antibody (1:1000; ABCAm, Eugene, OR), rabbit anti‐ASC antibody (1:1000, CST), rabbit anti-Fn14 antibody (1:1000, ABCAm), rabbit anti-β-actin antibody (1:7500, Signal way Antibody, College Park, MD, USA), and rabbit anti‐α‐tubulin antibody (1:7500, Servicebio, China). After incubation with peroxidase‐conjugated secondary antibodies (1:7500, Signal way Antibody) at room temperature for 1 h, the signal was developed using an ECL chemiluminescence kit (Millipore, USA). The band intensities were quantitated using Image Lab software and normalized to internal reference values accordingly.

Mouse anti-Tim23 was from Santa Cruz Biotechnology (CA, USA). Rabbit anti-Hsp60 antibody, FITC-conjugated anti-rabbit IgG antibody, and HRP-conjugated anti-mouse, rabbit, or goat IgG antibodies were purchased from Proteintech (Wuhan, China). Rabbit anti-β-actin, anti-LC3, anti-ERK, anti-p38, anti-p65, anti-phospho-ERK (Thr202/Tyr204), anti-phospho-p38 (Thr180/Tyr182), and anti-phospho-p65 (Ser536) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-IL-1β antibody was from R&D (Minneapolis, USA). Mouse anti-caspase-1 (p20) and mouse anti-Nlrp3 antibodies were purchased from Adipogen (Liestal, Switzerland).