B600020

Briefly, the sections were blocked with goat serum (C0265; Beyotime) at room temperature for 30 min. The sections were cotreated with primary antibody against TRPV2 (1 : 100; BS-10297R; Bioss) overnight at 4°C. Horseradish peroxidase-labeled anti-mouse IgG antibodies (ATPA00025Go; AtaGenix, Wuhan) were added to the sections and cultured at room temperature for 30 min. Then, the sections were colored by DAB for 5-10 min. Nuclei were counterstained with hematoxylin (B600020; Proteintech) for 3-5 min. After dehydration and transparency, the sections were sealed with neutral gum.

About 3 cm of small bowel tissue above 2 cm from the ileocecal area was fixed with 10% formalin, paraffin embedded, and sectioned. After dewaxing with xylene, a series of ethanol was used for hydration. The sections were stained with hematoxylin (B600020; Proteintech, China) for 5 min. After washing with alkaline PBS and bluing for 2 min, they were rinsed with running water for 3 min. Afterwards, they were stained with eosin (Sigma, USA) for 10 min and flash washed with distilled water. Neutral gums were used for coverslipping. The morphological and structural changes of intestinal mucosal epithelium were observed under an optical microscope (eclipse Ci-e; Nikon, Japan). According to the results of hematoxylin and eosin staining, the degree of intestinal injury was evaluated by Chiu's score [15 (link)].

Brain tissue specimen was fixed via 4% paraformaldehyde and embedded in paraffin. The section was cut into 4 μm. After xylene deparaffinization, hydration and antigen retrieval, the section was sealed through normal goat serum (C0265; Beyotime, Shanghai, China) lasting 20 min at room temperature. Then, the section was treated by primary antibodies against NLRP3 (1/100; NBP2-12446; Novus, USA) and IL-1β (1/50; Abs126104; absin, Beijing, China) at 37°C for 90 min and incubated by secondary antibody (1/100; SA00001-2/SA00001-1; Proteintech, Wuhan, China) lasting 30 min at room temperature. The color developing was presented via DAB reagent for 5 min. The nucleus was counterstained through hematoxylin (B600020; Proteintech, Wuhan, China) lasting 3 min as well as the section was washed by running water. After dehydration, the section was transparent and sealed with neutral gum. Images were acquired through an IX71 microscope (Olympus, Japan). The results were quantified with ImageJ software (version 1.48; National Institutes of Health).