Transmission electron microscopy

Huh-7 and SMMC7721 cells were exposed to iRFA treatment alone or iRFA treatment with 3-MA and then further incubated for 24 h. Next, the cells were fixed with 2.5% glutaraldehyde and then rinsed three times (30 min each) with 0.1 M PBS. Subsequently, the cells were fixed with 1% osmium tetroxide for 2 h and gradient-dehydrated in acetone, saturated, and embedded in epoxy 618. Semi-thin slices and then ultra-thin slices were prepared and stained with uranium and lead. Finally, autophagy was evaluated using transmission electron microscopy (Tecnai 10; Philips, Eindhoven, The Netherlands).