Gb111402

Colon tissues were formalin fixed overnight at room temperature and then embedded in paraffin. 5 µm sections were stained with hematoxylin and eosin (HE) for pathological analysis. The pathological score was assessed by the severity of inflammation, the degree of inflammatory involvement, and the degree of epithelial/crypt damage as previously reported.^{58 (link)} Calculate each parameter and sum to get the total score.
For immunohistochemistry, paraffin-embedded tissue sections were stained with anti-MUC2 antibody (GB14110, 1:1000, Servicebio, China), anti-ZO-1 antibody (GB111402, 1:1000, Servicebio, China) and visualized by DAB staining according to manufacturer’s instructions.
For immunofluorescence, paraffin-embedded tissues were stained with anti-F4/80 antibody (GB11027, 1:1000, Servicebio, China), anti-Il-10 antibody (GB11108, 1:1000, Servicebio, China) and anti-CD206 antibody (GB113497, 1:500, Servicebio, China). The slides were independently assessed by two researchers who were unaware of the nature of the samples.

Colon tissues were fixed in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining and Periodic acid Schiff (PAS) staining were conducted to assess morphological changes. The paraffin-embedded colon tissues were deparaffinized, rehydrated, retrieved, and antigen retrieval. Tissues were sealed with 3% bovine serum albumin and incubated with primary antibodies: anti-Muc2 antibody (Servicebio, GB14110, 1:500), anti-ZO1 antibody (Servicebio, GB111402, 1:500), anti-Occludin antibody (Servicebio, GB111401, 1:500), anti-Lgr5 (Bioss, bs-20747R,1:100). The slices were then incubated with secondary antibodies (Fdbio, FD0129, 1:500; Fdbio, FD0136,1:500). The images were scanned with Pannoramic Scanner (Pannoramic DESK, 3DHISTECH) and observed with Caseviewer C.V 2.3.

Mice were transcardially perfused with 0.1 M phosphate buffer (PB) followed by 4% paraformaldehyde (PFA) under deep anesthesia. The distal colons were removed, postfixed in 4% PFA and embedded with OCT. The sections (10 μm) were blocked with 10% normal goat serum in 0.05 M PBS and were incubated with one of the following primary antibodies at 4°C overnight: 1) rabbit anti-occludin (1:1500; GB111401; Servicebio, Shanghai, China); 2) rabbit anti-claudin-1 (1:500; GB11032; Servicebio); and 3) rabbit anti-ZO-1 (1:1500; GB111402; Servicebio). The following day, sections were incubated with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:1000; Molecular Probes-Invitrogen, Eugene, USA) at room temperature for 1 h. The sections were viewed under a fluorescence microscope (Leica DM2500; Leica, Wetzlar, Germany), and digital images were captured using Leica Application Suite version 4.3 (Leica). Integrated density was measured to evaluate fluorescent signals using ImageJ (
http://rsb.info.nih.gov/ij/).

The pre-processing was the same as the previous steps to obtain 5 µm-thick sections of colons. For the immunohistochemical analysis, the rabbit anti-Muc2 (GB11344, Servicebio, Wuhan, China) primary antibody was applied at 1:500, then stained with FITC-conjugated (GB25303, Sevicebio) anti-rabbit secondary antibody. For the immunofluorescence analysis, the rabbit anti-claudin-1 (GB11032, Servicebio) and -ZO-1 (GB111402, Servicebio) primary antibodies were applied at 1:500, then stained with the HRP-conjugated (GB23303, Servicebio) anti-rabbit IgG secondary antibody. All images were captured with a fluorescence microscope (Nikon, Tokyo, Japan).

For immunofluorescence staining, 4-μm paraffin sections were prepared, and, after dewaxing and hydration, the samples were incubated in 3% H₂O₂ for 15 min at room temperature. After three washes in PBS for 5 min each, the samples were incubated in 5% BSA at room temperature for 30 min. The first antibody of ZO-1 (GB111402, Servicebio) and occludin (GB111401, Servicebio) was incubated with the sections overnight at 4°C. The second fluorescent antibody was incubated with the sections at room temperature for 50 min. After rinsing with PBS and then staining with DAPI, the paraffin sections were screened by a scanner (Pannoramic DESK, P-MIDI, P250, 3D HISTECH). DAPI-stained nucleus was in blue; positive occludin protein expression was in red; and positive ZO-1 protein was in green.

Tissues were prepared for sections (5μm), stepwise (200μm) through the paraffin block. The slides were dehydrated by gradient alcohol and stained with HE. The colon tissue of epithelial injury, inflammatory infiltration, and dysplastic hyperplasia was evaluated by pathologists separately.
As described above for IHC, the slides were blocked with 5% BSA for 1 h and incubated with primary antibody against ZO-1 (GB111402, Servicebio, China) and occluding (27260-1-ap, Proteintech, China) overnight at 4°C followed by incubation with secondary antibody for 1 h at room temperature. The sections were stained with DAB and then counterstained with hematoxylin.
To stain immunofluorescence, the experiment was carried out as mentioned above until the antigen is retrieved by citric acid buffer (PH6.0) through the microwave. Then, the slides were incubated with primary antibody overnight at 4°C after blocking. The secondary antibody conjugated with Alexa Flour 488 or 594 was used to incubate with the slides for immunofluorescence and DAPI for nuclei. The stained specimens were scanned by the laser scanning confocal microscopy (Leica DMIRE2, Germany).