Rabbit anti nlrp3

Sodium hydrogen sulfide (NaHS, #161527) was purchased from Sigma-Aldrich. The normal human hepatocytes L02 cells (#CL-0111) were purchased from Procell. The content of IL-1β was determined using a commercial ELISA kit (Elabscience, #E-EL-H0149c). The primary antibodies, including rabbit anti-NLRP3 (#Ag26289, 1:500), rabbit anti-LC3 (#14600-1-AP, 1:1000) mouse anti-AMPK (#10929-2-AP, 1:1000) and rabbit anti-mTOR (#28273-1-AP, 1:1000) were purchased from Proteintech, rabbit anti-cleaved caspase-1 (#89332, 1:1000), rabbit anti-procaspase-1 (#24232, 1:1000), rabbit anti-Beclin-1 (#3495, 1:1000), mouse anti-β-actin (#3700, 1:2000) and rabbit anti-GAPDH (#8884, 1:2000) were purchased from Cell Signaling Technology.

Hippocampal neurons and brain tissue samples were collected for Western blot analysis. Protein samples were separated by 10% SDS–polyacrylamide gel electrophoresis gels and electrotransferred onto 0.22-μm polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) at RT for 1 h and then incubated with mouse anti-GPR120 (1:500; Santa Cruz, USA), mouse anti-Caspase-1 (1:500; Santa Cruz, USA), mouse anti-IL-1β (1:1000; CST, UK), mouse anti-IL-18 (1:500, Proteintech, China), rabbit anti-NLRP3(1:500; Proteintech, China), rabbit anti-ASC (Proteintech, China), rabbit anti-GAPDH (Proteintech, China) antibodies overnight at 4 °C. The blots were washed three times and incubated for 1 h with horseradish peroxidase HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (1:5000; Proteintech) in TBST. The blots were then washed in TBST, and bands were visualized using an enhanced chemiluminescence system (Advansta, USA) and the ChemiDoc Touch Imaging System (Bio-Rad). Relative protein levels were determined by normalization to the GAPDH signal using Image lab software (Bio-Rad, CA, USA).

With regard to the immunofluorescence method, myocardial tissue slices were incubated with the following primary antibodies: Rabbit anti-NLRP3 (1:100, Proteintech, 19771-1-AP, United States), anti-Caspase 1 (1:50, Proteintech, 22915-1-AP, United States), anti-IL-1β (1:200, Proteintech, 26048-1-AP, United States), anti-IL-18 (1:400, Proteintech, 10663-1-AP, China), anti-ASC (1:800, Cell Signaling, #67824, United States).
The slices were then washed and detected with appropriate Fluor dye, DyLight 488 (1:50, EarthOx, E032220-01, United States), as secondary antibodies followed by counterstaining with DAPI in the dark. Afterward, immunofluorescent signaling was observed with a fluorescence microscope (IX73 Olympus). Digital images and data were recorded and analyzed by applying ImageJ (NIH).