Ab240991

MH-S cell were placed on slides, fixed with 4% paraformaldehyde at room temperature for 30 min, then washed with PBS for 2–3 times. Placed the cell slides in 1% Triton x-100 (JISSKANG, China) for 10 min. After washing with PBS, the cell slides were blocked in 5% BSA for 1 h. Then the diluted anti-mouse caspase-11(ab240991, abcam, 1:100) was added and incubated overnight at 4 °C. On the second day, incubate the cell slides with the fluorescent secondary antibody (Beyotime, China) at 37 °C for 1 h under the dark environment. After rinsing, fluormount with DAPI (AntGene, China) was added to stain the nucleus. Finally, seal the tablet and observe the slides under the fluorescence microscope.

Proteins of cells and lung tissues were extracted by RIPA Lysis Buffer (CoWin Biosciences, China) with phenylmethanesulfonylfluoride (PMSF; 1:100, MilliporeSigma) and protease inhibitor cocktail (1:100, Servicebio, China) and quantified by BCA protein assay kit (ECOTOP, China) for SDS-PAGE electrophoresis. Separated proteins were transferred to a polyvinylidene difluoride membrane (PVDF). The PVDF membrane was incubated with first antibodies overnight at 4 ℃ after blocked with 5% skim milk for 1 h. Antibodies against caspase-11 (ab240991, abcam, 1:1000), Gsdmd (ab219800, abcam, 1:1000), NLRP3 (15101, Cell Signaling Technology, 1:1000) and HRP-conjugated secondary antibodies (Proteintech, 1: 10,000) were used.