Caspase 12

After euthanasia, the liver and pancreas were processed, including 10% neutral formaldehyde fixing, paraffin embedding, 5 µm thickness sectioning, and H&E staining [26 (link)]. The pathological changes were observed using a stereomicroscope (SMZ25, Nikon, Tokyo, Japan). For immunohistochemistry, the sections of the liver were antigen retrieval, blocked with goat serum (37 °C for 20 min), and then incubated with primary antibodies. The antibodies included AMPK (1:100, Beyotime, Shanghai, China), Phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK, 1:200, Bioss, Beijing, China), Dynamic-related protein 1 (DRP1, 1:100, Beyotime), Cytochrome c (CytC, 1:100, Proteintech, Chicago, IL, USA), caspase 12 (1:100, Bioss), Inositol-requiring enzyme-1 (IRE1, 1:100, Proteintech) and Mitofusion 1/2 (MFN1/2, 1:100, Proteintech). Subsequently, the slides were visualized using DAB and then observed under a stereomicroscope (SMZ25, Nikon).

Protein was abstracted from frozen intestinal tissue. Extracted protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose blotting membranes (Roche, CA, USA). The membranes were incubated with primary antibodies to Tubulin (Proteintech, wuhan, china), Grp78 (Bioworld, USA), Caspase-12 (Bioss, BeiJing, China), Gadd153 (Bioworld, USA), ATF4 (Santa Cruz, USA), P-elF2 (Bioeasy, shanghai, china) and P-PERK (Santa Cruz, USA), and horseradish-peroxidase–conjugated secondary antibodies for 2 hours, then developed with SuperSignal West Pico chemiluminescent Substrate Kit (Pierce, Rockford, USA), and finally analyzed with the Bio-Image Analysis System (Syngene, Frederick, MD, USA).