A549 cells were fixed with 4% paraformaldehyde for 15 min and then blocked for 30 min with 5% BSA. The cells were first treated with primary antibodies against ZO-1 and occludin overnight at 4 °C, followed by 1 h at room temperature with fluorescein isothiocyanate-conjugated secondary antibodies (Alexa Fluor 488 IgG, Beyotime). Samples were captured using a confocal microscope (Nikon, Japan).
Lung tissues were sectioned into 5-μm-thick slices, deparaffinized with xylene, and then dehydrated with an ethanol gradient. After thermal repair of the antigens, the samples were blocked with goat serum for 30 min at room temperature and then incubated overnight at 4 ℃ with primary antibodies against ZO-1 and occludin. The slides were incubated with fluorescein isothiocyanate-conjugated secondary antibodies (Alexa Fluor 488/Cy3 IgG, Beyotime) for 1 h at room temperature. The sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) (#D8417, Sigma-Aldrich) for 8 min, and the slices were sealed with a fluorescence decay-resistant medium. The samples were imaged with a fluorescence microscope (Nikon).
Lung tissues were sectioned into 5-μm-thick slices, deparaffinized with xylene, and then dehydrated with an ethanol gradient. After thermal repair of the antigens, the samples were blocked with goat serum for 30 min at room temperature and then incubated overnight at 4 ℃ with primary antibodies against ZO-1 and occludin. The slides were incubated with fluorescein isothiocyanate-conjugated secondary antibodies (Alexa Fluor 488/Cy3 IgG, Beyotime) for 1 h at room temperature. The sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) (#D8417, Sigma-Aldrich) for 8 min, and the slices were sealed with a fluorescence decay-resistant medium. The samples were imaged with a fluorescence microscope (Nikon).