Immunofluorescence Analysis of Mitochondrial Dynamics

The cells were washed twice with PBS, permeabilized in 0.1% Triton X-100, neutralized with NH₄Cl buffer and then per-meabilized using 0.05% saponin/PBS (pH 7.4) for 45 minutes. Subsequently, the samples were incubated overnight with the following primary antibodies: cyt-c (1:500; Abcam, #ab90529), Drp1 (1:1,000; Abcam, #ab56788) and Tom-20 (1:1,000; Abcam, #ab186735). Confocal immunofluorescence images were collected using the FV10-ASW 1.7 software and an Olympus IX81 microscope.24 The fluorescence intensity was calculated using the Image-Pro Plus 6.0 software. First, fluorescence pictures were converted to grayscale with the Image-Pro Plus 6.0 software. Then, the fluorescence intensities were separately recorded as the grayscale intensities. Mitochondria were observed in at least 100 cells, and the average length of the mitochondria was measured under an inverted microscope to quantify mitochondrial fragmentation (BX51; Olympus Corporation, Tokyo, Japan) as described in a previous study.25 (link) The experiments were performed in triplicate and repeated three times with similar results.26 (link)