Gb11032

The 5-µm-thick paraffin-embedded jejunal and ileal tissue sections were used for immunofluorescence. Heat-induced antigen retrieval was performed by autoclaving the sections for 10 minutes at 121°C in 10mM sodium citrate buffer (pH 6.0). The sections were blocked with 8% skim milk in TBST at 37°C for 40 minutes, and then immunostained using primary antibodies against ZO-1 (1:200, GB11195, rabbit; Servicebio), occludin (1:200, GB111401, rabbit; Servicebio), claudin-1 (1:200, GB11032, rabbit; Servicebio) at 4°C overnight. The sections were washed and incubated with secondary fluorescent antibodies at 37°C for 60 minutes. The secondary antibodies was CY3 goat anti-rabbit IgG (1:300; GB21303; Servicebio). Sections were mounted with Nikon DS-U3 with DAPI (G1012, Servicebio). Images were captured with an Nikon Eclipse C1 fluorescence microscope (Nikon, Tokyo, Japan).

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 colon tissues were fixed in 4% paraformaldehyde, embedded in paraffin, dewaxed in xylene and rehydrated in gradient alcohols. The tissue sections were retrieved in citrate buffer (pH 6.0) and incubated with primary antibodies (rabbit anti-occludin, 1:500, GB111401, Servicebio; rabbit anti- zonula occludens-1, 1:200, A0659, Abclonal; rabbit anti-claudin, 1:500, GB11032, Servicebio) overnight at 4 °C. Next, these sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:200, GB23303, Servicebio, China) at 25 °C for 2 h. After washing in PBS, the sections were counterstained with haematoxylin for 3 min, dehydrated and mounted. For quantification, five fields were randomly selected for each sample. The expression of occludin, ZO-1 or claudin protein was quantified by the histochemistry score (H-score) by selecting five regions on each slice. The average H-score values from the five regions were statistically analysed to determine the overall percentage of occludin, ZO-1 or claudin protein-positive area across the four groups. H-score = ∑ (pi × i) = (percentage of weak intensity × 1) + (percentage of moderate intensity × 2) + (percentage of strong intensity × 3)^{54 (link),55 (link)}.

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).