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.