Transmission electron microscopy

Electron microscopy was performed to examine the morphology of the mitochondria in HUVECs. Briefly, after incubating as described above, cells were collected and prefixed with 2.5 % w/v glutaraldehyde for 2 h, rinsed three times in 0.1 mol/l PBS (pH 7.4), and post-fixed for 2 h in 1 % w/v osmium tetroxide at −4 °C. The fixed cells were dehydrated through an ethanol series then completely dehydrated in absolute ethanol. Cells were detached using propylene oxide and infiltrated with Spurr low-viscosity embedding medium (Wuhan Boshide Biological Technology Co., Wuhan, China). Sections were cut using an ultramicrotome with a diamond knife, and stained with uranyl acetate and lead citrate. Cells were observed using a transmission electron microscope (JEM-1230, Olympus Corporation, Tokyo, Japan).