4.3. Immunofluorescence and Analysis of Mitochondrial Morphology

Cells were cultured on glass coverslips for 24–48 h, followed by fixation with 4% paraformaldehyde for 10 min and permeabilization with 0.1% Triton X-100 in PBS for another 10 min at room temperature. After blocking with 3% BSA for 30 min, cells were stained with mouse anti-Tom20 monoclonal antibody (Santa Cruz, Dallas, TX, USA, sc-17764) and then incubated with the Goat Anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Invitrogen, Waltham, MA, USA, A-11003). Following this procedure, cell nuclei were counterstained with DAPI (Thermo Fisher Scientific, Waltham, MA, USA, D1306), and images were acquired under a Leica confocal microscope (Buffalo Grove, IL, USA, TCS SP5) with appropriate excitation and emission filter pairs. For mitochondrial morphology analysis, aspect ratio (an index of mitochondrial branch length) means the ratio between the bigger (major) and the smaller (minor) side for each mitochondrial fragment. The images were processed to maximize the signal/background ratio using the automatic adjustment of brightness/contrast of the ImageJ software. Finally, the major and the minor of each fragment were determined using the “analyse particles” function of ImageJ. Analyses were performed on at least 50 cells for each condition [75].