Primary antibodies against zo1

Total proteins were extracted from the jejunum tissue samples and IPEC-J2 cells using the RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing protease inhibitors. The protein concentrations were determined using the BCA protein assay kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). All of the extracted proteins were diluted with the SDS loading buffer and boiled at 95 °C for 10 min. A total of 40 µg extracted protein were separated by 12% SDS-PAGE gel electrophoresis and then transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk for at least 1 h at room temperature, and then were incubated with the primary antibodies against ZO-1, Occludin, Claudin-1, and β-actin (Proteintech, Chicago, IL, USA) overnight at 4 °C. The membranes were washed four times with 1 × Tris-buffered saline-Tween (TBST) for 10 min and incubated with the anti-mouse (or rabbit) IgG HRP-conjugated secondary antibody (1:3000; Cell Signaling Technology, Danvers, USA) for 1 h. After washing with 1 × TBST three times, the target protein bands were visualized with the electrochemiluminescence visualized system (Tanon, Shanghai, China). Band intensities were quantified using the ImageJ version 1.51 software [20 (link)], and all results were expressed as the target protein/β-actin protein ratio.

Immunofluorescent staining was performed to evaluate the expression of tight junction proteins, including claudin-1, Zonula Occludin (ZO1), and Occludin. A total of 5 µm paraffin-embedded colon tissue was cut and placed on a positive charge slide. Then kept in xylene for 10 min twice to deparaffinized and rehydrate in a series of gradient-decreasing concentrations of ethanol. Slides were treated in citrate buffer for antigen retrieval in a microwave oven at 350 watts for 15 min. Blocking reagents were used to block the tissue sections of slides for 30 min. After blocking, slides were washed with PBS three times for 5 min and incubated overnight with primary antibodies against ZO1, Occludin, and Claudin (proteintech, Wuhan, China) at 4 °C. After washing, the slides were then incubated with fluorescein (FITC)-conjugated Affinipure goat anti-rabbit (proteintech) secondary antibodies for one hour, and DAPI was used for 5 min to stain the nucleus. Finlay, the slides were examined under a microscope, and images were captured.

Immunofluorescent staining was performed to evaluate the expression of tight junction proteins, including claudin-1, Zonula Occludin (ZO1), and Occludin. A total of 5 µm paraffin-embedded colon tissue was cut and placed on a positive charge slide. Then kept in xylene for 10 min twice to deparaffinized and rehydrate in a series of gradient-decreasing concentrations of ethanol. Slides were treated in citrate buffer for antigen retrieval in a microwave oven at 350 watts for 15 min. Blocking reagents were used to block the tissue sections of slides for 30 min. After blocking, slides were washed with PBS three times for 5 min and incubated overnight with primary antibodies against ZO1, Occludin, and Claudin (proteintech, Wuhan, China) at 4 °C. After washing, the slides were then incubated with fluorescein (FITC)-conjugated Affinipure goat anti-rabbit (proteintech) secondary antibodies for one hour, and DAPI was used for 5 min to stain the nucleus. Finlay, the slides were examined under a microscope, and images were captured.