Rabbit anti caspase 1

Brain and gut tissues were collected, flash-frozen, and stored at −80 °C. Methods for SDS-PAGE electrophoresis and Western blotting were adapted from our previous work [43 (link)]. The following primary antibodies were selected: mouse anti-NLRP3 (#AG-20B-0014-C100; Adipogen, San Diego, CA, USA), rabbit anti-Caspase-1 (#A0964; Abclonal, Woburn, MA, USA), or mouse anti-GAPDH (#AM4300; ThermoFisher Scientific, Waltham, MA, USA). Donkey anti-mouse or goat anti-rabbit HRP-labeled IgGs (Vector Laboratories, Burlingame, CA, USA) were used as secondary antibodies. Bands were detected by enhanced chemiluminescence using the SuperSignal™ West Femto substrate (Thermo-Fisher Scientific, Waltham, MA, USA). Membranes were imaged with the Odyssey Fc imaging system (Li-Cor, Lincoln, NE, USA). Signal density was quantified using the complementary software, Image Studio Lite (Li-Cor, Lincoln NE, USA), with the relative expression of NLRP3 and Caspase-1, normalized to endogenous GAPDH levels.
Two-way analysis of variance (ANOVA) was used to determine the effects of genotype and age on biochemical and morphological measures using GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). Values of p < 0.05 were considered statistically significant, and data are expressed as mean ± SEM.

For primary microglia, after indicated treatment, cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized by 0.5% Triton X-100 for 30 min. Following blocking with 5% BSA for 1 h, cells were incubated with indicated primary antibodies overnight at 4°C. After three washes with PBS, cells were stained with DayLight 488/CY3-conjugated secondary antibodies (1:400, Abbkine) for 1 h. DAPI was used to stain nuclei. Images were recorded by Leica confocal microscopy. For brain tissue, double immunofluorescence staining was performed on formalin-fixed, paraffin-embedded brain sections. After blocking with 5% BSA, 4 μm thick paraffin brain slices were incubated with rabbit anti-Caspase-1 (1:200, Abclonal), rabbit anti-GSDMD (1:200, Abclonal), or mouse anti-Iba-1 (1:200, Abcam) antibodies overnight at 4°C. Then, the slices were incubated with CY3-conjugated duck anti-rabbit or DayLight 488-conjugated duck anti-mouse secondary antibodies (1:400, Abbkine) for 1 h at 37 °C. After washing with PBS, DAPI was used to label the nuclei. The number of double-positive cells was detected under a fluorescence microscope.