Anti claudin 1 antibody

Escherichia coli LPS (055:B5) was purchased from Sigma-Aldrich (St. Louis, MO, USA). DHM was purchased from Shanghai Winherb Medical Technology Co., Ltd. (Shanghai, China; CAS No. 27200-12-0) and was purified (purity > 98.0%) from Chinese Rattan tea by high performance liquid chromatography. Anti-claudin-1 antibody was purchased from ABclonal Technology (Wuhan, China). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Huamei Biological Engineering Co., Ltd. (Wuhan, China). Anti-bax, anti-bcl-2, anti-caspase-3, anti-occludin, anti-ZO-1, anti-TLR4, anti-NF-κB p65 and anti-phospho-p65 antibodies, HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were purchased from Bioss Biotech Co. Ltd. (Beijing, China).

The distribution of the tight-junction protein (claudin-1) in the IPEC-J2 cells was determined by an immunofluorescence analysis. Briefly, the IPEC–J2 cells were seeded on cover-slides treated with poly(L-lysine) (Biosharp, Beijing, China) and placed in 12-well plates for 12 h to reach 70% confluence. The cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 30 min, and then they were permeabilized with 0.5% Triton X-100 buffer (Beyotime, Shanghai, China) at room temperature for 20 min. Thereafter, the IPEC–J2 cells were incubated with anti-claudin-1 antibody (dilution 1:500; ABclonal, Wuhan, China) for 1 h at room temperature and then incubated with fluorescein conjugated goat anti-rabbit IgG (H + L) antibody (dilution 1:500; Proteintech, Wuhan, China) in the dark for 1 h. The cell nuclei were stained with 4′,6-Diamidino-2-Phenylindole (DAPI, Beyotime, Shanghai, China) solution. The slides were visualized under a laser scanning confocal microscope (Zeiss, LSM 700; Oberkochen, Germany).