Cleaved il 1β

The colon tissues were homogenized with the ice−cold RIPA buffer containing protease inhibitors (Beyotime Biotechnology, Shanghai, China). The colon proteins were collected after centrifugation at 14,000× g for 5 min and determined using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated by 4–12% SDS−PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking at room temperature with a commercial blocking buffer (Beyotime Biotechnology, Shanghai, China), membranes were incubated overnight at 4 °C with the following primary antibodies: Caspase 3, B cell lymphoma2 (Bcl2), Bcl2 associated X (Bax), cleaved Caspase 3 P17, cleaved Caspase 1 P20, and cleaved IL−1β, which were purchased from Affinity Biosciences (Cincinnati, OH, USA); Occludin, Claudin 1, ZO−1, ACS, NLPR3, Caspase 1, IL−1β, GSDMD, and β−actin, which were purchased from Proteintech (Wuhan, Hubei, China). Then, membranes were incubated with HRP−conjugated secondary antibody for 2–2.5 h at room temperature. Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Wilmington, DE, USA) and detected by the ChemiDoc™ imaging system (BIO−RAD, Hercules, CA, USA). The results were quantified using Image J software and normalized to β−actin.

For zebrafish intestinal immunohistochemistry, OCT (Sakura Finetek, Japan)-embedded frozen sections were fixed using anhydrous methanol (Servicebio, China) for 20 min. Antigen retrieval was carried out using 10 mM sodium citrate (pH 6) in a microwave for 15 min. Endogenous peroxidases were quenched with 3% H₂O₂ for 25 min. All slides were blocked with 10% nonspecific antigen with normal rabbit serum (BosterBio, AR1010, USA) for 30 min at room temperature and then incubated with 1:50 diluted cleaved-IL-1β (Affinity Biosciences, AF4006, USA) antibody overnight at 4°C. After that, slides were incubated with 1:200 diluted horseradish peroxidase-conjugated secondary antibodies (Serivcebio, GB23303, China) at room temperature for 30 min and developed with 3,3’-diaminobenzidine (DAB) solution (DAKO, K5007, Denmark). Slides were counterstained with hematoxylin. Images of stained slides were processed by Nikon Eclipse TS100 (Nikon, Japan).

Mdivi-1(mitochondrial division inhibitor-1) and TTC (2,3,5-triphenyltetrazolium chloride) were purchased from Sigma Aldrich (St. Louis, Missouri, MO, USA); BSA (bovine serum protein) was obtained from BOSTER (Boster Biological Technology, Wuhan, China, AR1006). Blots were incubated with antibodies against ATF4 (Proteintech, Rosemont, Pennsylvania, USA, 10835-1-AP), Parkin (CST, Boston, Massachusetts, USA, #4211), TOM20 (Proteintech, USA, 11802-1-AP), COX4I1 (Proteintech, USA, 60251-1-Ig), NLRP3 inflammasome (Affinity, Cincinnati, Ohio, USA, DF7438), pro-caspase-1 (ABclonal, Wuhan, China, A0964), cleaved caspase-1 (CST, USA, #67314), pro-IL-1β (ABclonal, China, A11370), cleaved IL-1β (Affinity, USA, AF4006), pro-IL-18 (Proteintech, USA, 10663-1-AP), cleaved IL-18 (R&D Systems, Minneapolis, Minnesota, USA, AF521) and β-actin (ABclonal, China, AC004), followed by the secondary antibodies conjugated to horseradish peroxidase anti-rabbit IgG (H + L) (ABclonal, China, AS014) and anti-mouse IgG (H + L) (ABclonal, China, AS003).
The primary antibodies used for the immunofluorescence analysis were against TOM20 (Proteintech, USA, 11802-1-AP) and COX4I1 proteins (Proteintech, USA, 60251-1-Ig). Brain sections were then incubated with the secondary antibodies goat anti-rabbit DyLight 488 (Abbkine, green, A23240) and goat anti-mouse DyLight 549 (Abbkine, red, A23310).