Paraffin-embedded sections of the ileum and gastric antrum were dewaxed, rehydrated, and subjected to antigen heat retrieval in citrate buffer followed by blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature. Next, sections were incubated with rabbit anti-NLRP3 (dilution 1 : 1500; Abcam, Cambridge, UK), anti-ASC (1 : 1000; Abcam, Cambridge, UK), and anti-caspase-1 (dilution 1 : 500; Santa Cruz Biotechnology, CA, USA) primary antibodies overnight at 4°C. After washing with PBS 3 times, slides were stained with Cy3 conjugated goat anti-rabbit IgG, FITC conjugated goat anti-rabbit IgG, or FITC conjugated goat anti-mouse IgG (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) secondary antibodies for 1 h at room temperature. Sections were washed three times, and nuclei were stained with 4′,6-diamidino-2- phenylindole (DAPI) for 10 min at room temperature. Images were captured using a fluorescence microscope (Eclipse C1, Nikon, Tokyo, Japan). For colocalization analysis, all sections were taken randomly and analyzed using ImageJ software, and the summarized colocalization efficiency data were represented by Pearson's correlation coefficient (PCC) [36 (link)]. The fluorescence intensity of caspase-1 in each group was also measured by ImageJ software.
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