Ab201792

Immunohistochemistry (IHC) staining was performed according to a previous method [13 (link), 20 ]. Serial paraffin sections were subjected to antigen retrieval, incubation in antigen retrieval solution for 20 min, inactivation with endogenous peroxidase (3% H₂O₂), and blocked in goat serum for 1 h. Sections were then incubated with primary antibodies against CD3 (SC-20047, Santa Cruz Biotechnology Inc., Dallas, TX, USA), F4/80 (SC-377009, Santa Cruz Biotechnology Inc.), IL-6 (GeneTex, Santa Cruz Biotechnology Inc.), IL-1β (ab9722, Abcam), TNF-α (ab183218, Abcam), p-p65 (Ser536) (#3033, Cell Signaling Technology), p16 (ab211542, Abcam), NLRC4 (ab201792, Abcam), ITGAM (ab133357, Abcam), p19 (ab80, Abcam), Collagen I (14695-1-AP, Proteintech), Collagen III (22734-1-AP, Proteintech), α-SMA (14395-1-AP, Proteintech), and β-galactosidase (15518-1-AP, Proteintech). After washing, sections were incubated with a secondary antibody for 1 h, and processed using the SABC-POD kit (SA2001, Boster, China). Then, sections were counterstained with Hematoxylin and fixed with biomount medium. Hematoxylin and Shandon Instant Eosin (Solarbio Co., Ltd.) were used to determine cell infiltration. Masson's trichrome staining (Sigma-Aldrich®) was used to assess collagen deposition.

Total protein was extracted from the cells by radioimmunoprecipitation assay (RIPA) lysate (AWB0136, Abiowell, China). Then, the protein was transferred to the polyvinylidene fluoride membrane after 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) treatment. The membrane was sealed with 5% skim milk (AWB0004, Abiowell) at room temperature for 2 h. AIM2 (1:1,500, 20590-1-AP, proteintech, Chicago, IL, USA), NLRC4 (1:1,000, ab201792, abcam, Cambridge, UK), NLRP3 (1:1,000, 19771-1-AP, proteintech), GSDMD-N (1:1,000, ab215203, abcam), ASC (1:2,000, 10500-1-AP, proteintech), caspase-1 (1:1,000, ab179515, abcam), IL-18 (1:8,000, 10663-1-AP, proteintech), IL-1β (1:1,000, 16806-1-AP, proteintech), and β-actin (1:5,000, 66009-1-Ig, proteintech) were incubated with the membrane at 4°C overnight. Then, the corresponding secondary antibodies were incubated with the membrane at room temperature for 2 h. The membrane was incubated with SuperECL Plus (AWB0005, abiowell), and then the protein bands were visualized by a chemiluminescence imaging system (ChemiScope 6100, Clinx, Shanghai, China).

Proteins were extracted using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Beyotime, China). Samples were homogenized on ice, centrifuged for supernatant at 12,000 g for 15 min at 4°C and heated to 100°C for 5 min. Protein extracts resuspended in sample loading buffer were separated by electrophoresis through 12% polyacrylamide gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk (Sangon Biotech Shanghai Co.,Ltd., China), membranes were incubated with primary antibodies anti-LC3 (4108S, CST, USA; 1: 1,000 dilution), anti-Beclin 1 (3738, CST, USA; 1: 1,000 dilution), anti-NLRP3 (15101S, CST, USA; 1: 1,000 dilution), anti-NLRC4 (ab201792, abcam, UK; 1: 1,000 dilution), anti-GAPDH (BA2913, Boster, China; 1: 1,000 dilution), anti-Tubulin (AF1216, Beyotime, China; 1: 1,000 dilution) and anti-Histone H3 (ab194681, abcam, UK; 1: 1,000 dilution) overnight at 4°C. Membranes were then washed and incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (A0208, Beyotime, China; 1: 3,000 dilution) for 1 h at room temperature. Proteins were visualized using ECL luminescence reagent (Meilunbio, China). The gray-scale values of the bands were determined by Image J launcher broken symmetry software program (National Institutes of Health, Bethesda, MD, USA).