Occludin antibody

The protein abundances of occludin and claudin-3 in jejunum and ileum were detected by immuno-histochemical analysis as described by previous study [34 (link)]. Briefly, slides were blocked with 5% bovine serum albumin (BSA), and then incubated with occludin antibody (1:500; Abcam; Cambridge, UK) and claudin-3 (1:150; Abcam; Cambridge, UK) overnight at 4 °C, washed three times for 5 min in phosphate buffer saline (PBS) (pH 7.4), and then incubated with secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:500; Wuhan service bio technology; Wuhan, China) for 50 min in the dark. Cell nucleus were stained with 40, 6-diamidino-2-phenylindole (DAPI) for 10 min and were washed with PBS (pH 7.4) three times for 5 min per wash, then treated with a self-fluorescence quenching agent for 5 min. After sealing, images were obtained under a fluorescence microscope (NIKON ECLIPSE C1; Nikon Corporation; Tokyo, Japan).

The total protein was extracted from the ileal tissue of mice using RIPA Lysis buffer (Seven Biotech; China, Beijing) according to the manufacturer’s instructions. The concentrations of the protein samples were determined using the BCA protein assay kit (Beyotime, Shang hai, China). Equal protein was separated on 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore, Billerica, MA). The membranes were incubated with primary antibodies: ZO3 (Abcam, UK; 1:1,000), occludin antibody (Abcam, UK; 1:1,000), and β-actin (Beyotime; China; 1:1,000) at a room temperature for 4 h, followed by incubation with the secondary Horseradish Peroxidase (HRP)-labeled antibody (Beyotime; China; 1:2,000). The optical density of specific bands was detected using an electrochemiluminescence detection system. The relative expression of protein level was quantified by densitometry using the Image J software (NIH, Bethesda, MD).

DSS (AppliChem, Lot#：A3261‐0250 Germany); the six medicinal herbs of QBD raw powder, as shown in Table 1 (Pharmacy Department, The Second Hospital affiliated to Shanxi University of TCM, Xianyang, China) were decoting up 150 mL by traditional decocting method for enema； Mesalazine Enemas (V Vifor AG Zweigniederlassung Medichemie Ettingen， H20150127, Switzerland); faecal occult blood (FOB) (Baso Diagnostics Inc， Zhuhai， china); NF‐κB p65 antibody (Cell Signaling, #8242, Danvers, MA, USA); Phospho‐NF‐κB p65 antibody (Cell Signaling, #3039); p44/42 MAPK (ERK1/2) (antibody (Cell Signaling，#4695); Phospho‐p44/42 MAPK (ERK1/2) antibody (Cell Signaling, #4370); MMP‐9 antibody (Merck, AB19016, Burlington, MA, USA); Ki67 antibody (Merck, AB9260); Cleaved Caspase‐3 antibody (Cell Signaling, #9664); Caspase‐3 antibody (Cell Signaling, #9662); β‐Actin antibody (Cell Signaling,# 4970) Notch1 antibody (Cell Signaling, #3608s); ZO‐1 antibody (abcam, ab96587, Cambridge, MA, USA); Occludin antibody (abcam, ab 21632); claudin‐1 antibody (abcam, ab15098); primer synthesis (Takara Bio Inc, Dalian, China).