Transmission electron microscopy

4×10⁶ cells were collected (n=3 for each sample) and fixed with 2.5 per cent glutaraldehyde for 12 hours, followed by 1 per cent osmium tetroxide for 1 hour. Samples were dehydrated with 50 per cent, 70 per cent, 90 per cent and 100 per cent acetone, and embedded in paraffin for 12 hours, followed by overnight incubation at 60°C. Samples were cut at 50–100 nm thickness and stained with 3 per cent uranyl acetate and lead nitrate. All specimens were examined with the HT-7700 transmission electron microscope (TEM; Hitachi, Tokyo, Japan). Autophagy and apoptosis were analyzed based on cell organelle structures, quantity and ratio of apoptotic cell and autophagic vacuoles.