Transmission electron microscopy of autophagosomes

The cells were fixed in 2.5% glutaraldehyde at 4°C overnight, and then washed three times with cold PBS. After fixing in 1% osmic acid at 4°C for 1 h, and dehydration with gradient alcohol and acetone, the cells were embedded in epoxy resin overnight at room temperature, and placed in an oven at 60°C for 2 h. 60-nm sections were cut from the blocks, and then double-stained with 2% uranyl acetate and lead citrate at room temperature for 5–10 min. The autophagosomes were observed under a transmission electron microscope (HT7700; Hitachi, Ltd.).