Anti caspase 1

Immunofluorescence staining was performed on both lung tissue and cellular slices through routine operation. For tissue staining, after dewaxing, rehydrating, antigen repairing, and blocking, paraffin-embedded tissue slices were incubated with diluted primary antibodies of anti-F4/80 (#GB11027, Servicebio), anti-Cytokeratin 7 (CK7, #GB11225, Servicebio), and/or anti-ADAR1 (#SC-73408, Santa Cruz), anti-cleaved-GSDMD (#AF4013, Affinity, Jiangsu, China), TUNEL (#GDP1043, Servicebio), anti-iNOS (#GB11119, Servicebio), or anti-CD163 (#GB11340-1, Servicebio) at 4 °C overnight, followed by incubation with their corresponding secondary antibodies at room temperature for 1 h in the dark. Between double staining procedures, 488-TSA (Servicebio) incubation and reantigen repair were conducted. DAPI (#G1012, Servicebio) was used for nuclear counterstaining. The slices were mounted with antifluorescence quenching sealing reagents (Boster, Wuhan, China) and captured under a fluorescence microscope (Life Technologies, Carlsbad, USA). For cellular staining, the primary antibodies used were anti-cleaved GSDMD (#AF4013, Affinity) and anti-caspase-1 (#GB11383, Servicebio). Mean fluorescence intensity (MFI) in a random field of view was measured using ImageJ software.

Protein from cells was extracted by rotor and radio immunoprecipitation assay (RIPA) lysis buffer. Equal amounts of protein were divided by SDS-PAGE in a 12% gel and transferred to a nitrocellulose membrane. The proteins were analyzed by an optimized chemiluminescence system based on the manufacturer's instructions. We incubated the membranes overnight with the primary antibodies: anti-Caspase 1 (Servicebio, Wuhan, China, GB11383); anti-CytoC (Servicebio, Wuhan, China, GB11080); anti-Bax (Servicebio, Wuhan, China, GB11690); anti-GAPDH (Affinity, Changzhou, China, AF7021); and anti-Bcl-2 (Affinity, Changzhou, China, AFfirm063, BF9103).

For immunoblotting at 24 hours after ROSC, the following antibodies were used: anti-Bak (rabbit, Servicebio), anti-caspase 3 (rabbit, Servicebio), anti-gasdermin D (GSDMD) (mouse, Santa Cruz), anti-caspase 1 (rabbit, Servicebio), anti-brain-derived neurotrophic factor (BDNF) (rabbit, Servicebio), anti-Drp1 (rabbit, Signalway Antibody), and anti-phospho-Drp1 (ser616) (rabbit, Signalway Antibody). Immunoreactivity was semiquantitatively calculated by Image J software (National Institute of Health, United States).