2.6. Immunofluorescent staining and mitophagy detection

Cells were first fixed with 4% paraformaldehyde for 30 min at room temperature. After incubation with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, the samples were treated with the primary antibodies at 4 °C overnight. After the slides were washed with PBS and then incubated with secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min. Nuclei were stained using DAPI. Images were observed with fluorescence microscopy (Olympus BX-61). The primary antibodies used in the present study were as follows: p-ERK (1:1000, Abcam, #ab176660), Bnip3 (1:1000, Cell Signalling Technology, #44060), Tom20 (mitochondria marker, 1:1000, Abcam, #ab186735), LAMP1 (lysosome marker, 1:1000, Abcam, #ab24170), cyt-c (1:1000; Abcam; #ab90529). Mitophagy is the result of fusion between mitochondria and lysosome. The green mitochondria locate with red lysosome would generate the orange mitophagy. Then, the number of orange dot was measured to quantify the number of mitophagy.