2.5 Cell immunofluorescence tests
A coverslip was placed into a 24-well plate and endothelial cell suspension was added onto the coverslip (8 ×104cells/well). When the cells had reached 90% confluency, the four groups were treated with different stimulatory factors for 48 hours. Then, the cells were washed three times with PBS, fixed in 4% paraformaldehyde for 30 min, and then washed three times with PBS. The cells were then sealed with sealing fluid (1x PBS/5% normal serum/0.3% TritonX-100) for 1h. Next, VE-cadherin antibody (1:100) and TRITC-labeled goat anti-rabbit IgG (1:200) were diluted with the same sealing fluid. Then, we added 300μl of diluted VE-cadherin antibody to each well and incubated at 4℃ overnight. Then, the cells were washed with PBS. Next, the cells were incubated with diluted fluorescent secondary antibody for 1h at room temperature without light, and then washed three times with PBS. Then, an anti-fluorescence quenching sealing solution (including DAPI) was used to seal the coverslip. Images were then acquired by laser confocal microscopy.