Immunofluorescence assessment
The effect of diosmetin on expression of ZO-1 was examined by means of immunofluorescence staining in vivo and in vitro. The tissue slices or formalin-fixed cells were incubated with anti-ZO-1 antibodies for overnight at 4 ℃. Following, washed three times with PBS, fluorescein-conjugated secondary antibody was incubated for 1 h at 37℃. The cell nuclei were stained with DAPI (5.0 μg/mL). Finally, the samples were photographed by using a confocal microscope (Nikon, Japan), with 400 × magnification.