Df7504

The amylase (MM-1018M1) and lipase (MM-1157M1) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Jiangsu MMBio Industry Co., Ltd (Yancheng, China). Tumor necrosis factor-α (TNF-α) (KE10002), IL-1β (KE10003) and IL-6 (KE10007) ELISA kits were purchased from Proteintech Group. Primary antibodies against the following proteins were purchased: rabbit anti-occludin (df7504, Affinity, 1/1000), rabbit anti-ZO-1 (af5145, Affinity, 1/500), rabbit anti-claudin-4 (af5350, Affinity, 1/500), rabbit anti-ASC (bs-6741R, Bioss, 1/500), rabbit anti-NLRP3 (bs-10021R, Bioss, 1/500) and rabbit anti-caspase1 (22915-1-ap, Proteintech, 1/500).

The paraffin sections were recovered by heating to 98°C in 10 mM citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase was blocked with 10% (v/v) H₂O₂ for 30 min. Non-specific antigens were blocked with serum at room temperature (20–25 °C) for 30 min. The paraffin sections were incubated with rabbit anti-occludin (1:100; DF7504; Affinity Biosciences Ltd., Liyang, China), rabbit anti-claudin-1 (1:100; AF0127; Affinity Biosciences Ltd., Liyang, China), and rabbit anti-ZO-1 (1:100; AF5145; Affinity Biosciences Ltd., Liyang, China) primary antibodies at 4 °C overnight and then treated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibodies (1:200; GB23303; Wuhan Servicebio Technology Co., Ltd., Wuhan, China). Photographic images were acquired under an OLYMPUS microscope and analyzed with ImagePro Plus software (Media Cybernetics, Rockville, MD, United States).

Tissue and cell lysates were separated, transferred to 0.45 μm PVDF membranes (Millipore, America), blocked and incubated in primary antibodies (metallothionein 2, MT2, 1 : 200, Immunoclone, America, ICA868Hu01). BCL2(1 : 1000, 15071), cleaved-caspase 3 (1 : 1000, 9661S), SOD2(1 : 1000, 13194), CAT (1 : 1000, 12980) (CST, America). cytochrome C (1 : 500, Abcam, Britain, ab133504). Bax (1 : 1000, 60267-1-Ig), VDAC1(1 : 1000, 66345-1-Ig) (Proteintech, Wuhan, China), malondialdehyde (MDA) (1 : 1000, Novus, America, NBP2-59366). claudin 5 (1 : 1000, #AF5216), ZO1 (1 : 1000, #AF5145), occludin (1 : 1000, #DF7504) (Affinity, Jiangsu, China), and β-actin (1 : 800, Boster, Wuhan, China, BA2305), and followed by incubation in HRP-conjugated secondary antibodies (Proteintech, Wuhan, China, SA00001-1 and SA00001-2). The membranes were visualized by the ECL system (Thermo, America, 32132), and the quantification of the band intensity were calculated by the ImageJ software [14 , 15 (link)].

Immunohistochemistry and immunofluorescence assay was used to identify the expression of E-cadherin, occludin, ZO-1, and MUC2. Paraffin-embedded colon sections were dewaxed and rehydrated before blocking to avoid endogenous peroxidase activity by 3% H₂O₂. For immunohistochemistry detection, colonic sections were incubated with primary antibodies against E-cadherin (Affinity, AF5145), occludin (Affinity, DF7504), ZO-1 (Affinity, AF5145), and MUC2 (Affinity, DF8390) overnight at 4°C, followed by incubating with streptavidin-horseradish peroxidase. The images were captured with a biological microscope (Olympus, CX21FS1). For immunofluorescent detection, the slices were treated overnight at 4°C with primary antibodies E-cadherin, occludin, and ZO-1. After washing with PBS three times to remove the extensive antibodies, the sections were incubated with secondary antibodies in the dark for an hour at room temperature. Lastly, the slides were incubated with DAPI solution (Wuhan Google Biotechnology Co., Ltd.), covered with a mounting medium, and examined under a fluorescence microscope (Nikon, Japan).

The protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit, and the samples were supplemented with loading buffer for western blot analysis. The proteins were separated by SDS-PAGE and subsequently transferred to PVDF membranes. After 1 h of blocking, the membranes were then incubated overnight at 4°C with specific primary antibodies, including anti-Claudin-1 (1:500; Ab15098; Abcam), anti-Occludin (1:1,000; Df7504; Affinity), ZO-1 (1:1000; Af5145; Affinity), and anti-β-actin (BM0627). Then the samples were incubated with the appropriate secondary antibody at room temperature for 1 h. Following the removal of the secondary antibody by washing, the protein bands were visualized using an ECL hypersensitive luminescent solution (Thermo Fisher, United States).

Brain tissues were fixed and then stabilized in 0.5% Triton X-100. After blocking with blocking buffer, the tissues were incubated with occludin (DF7504, Affinity, Jiangsu, China), LC3 (AF5402, Affinity, Jiangsu, China), and zonula occludens-1 (ZO-1) (21773-1-AP, proteintech, Shanghai, China) overnight at 4 °C. The tissues were then incubated with an anti-rabbit antibody and counterstained with DAPI. The cells were observed under a microscope.