Immunofluorescence Analysis of Cellular Organelles

HepG2 cells were fixed in 4% paraformaldehyde at room temperature for 30 min and then permeabilized using 0.05% Triton X-100 at 4 °C for 4 h [52 (link)]. Then, cells were washed with cold PBS to remove free Triton X-100. Subsequently, samples were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the present study were as follows: Cyt-c (1:500; Abcam; #ab90529), F-actin (1:500, Abcam, #ab205), p-JNK (1:500; Cell Signaling Technology, #9251) and Tom20 (1:500, Abcam, #ab186735). Tom20 was used to observe mitochondrial fission according to previous study [53 (link)]. Immunofluorescence was assessed under an Olympus IX81 microscope using FV10-ASW 1.7 software [54 (link)].