Immunofluorescent Analysis of Mitochondrial Morphology

Cells plated on coverslips in 12-well plates and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% v/v Triton X-100/1xPBS (10 min), and blocked with 10% normal goat serum prior to incubation with primary antibodies overnight. Rabbit anti-ki67 (Millipore, 1:200), rabbit anti-Tom20 (Thermofisher, 1:1000), mouse anti-cytochrome C (BD, 1:500), rabbit anti-APP (Cell sig, 1:200), mouse anti-P62 (abcam, 1:500) and mouse anti-Tom20 (Santa Cruz, 1:100). Cells were then labeled with appropriate secondary antibody raised in goat (Invitrogen). Images were taken with the LSM800 (Zeiss) confocal microscope by using the 40×/0.5 EC Plan-Neofluar objective or the 63×/1.4 Oil Plan-Apochromat objective. Excitation and acquisition parameters were constrained across all paired comparisons. For fluorescent quantification, morphometric measurements of images were performed using Image-Pro Plus software (MediaCybernetics). Mitochondrial morphology quantification was conducted by the Mito-Morphology Macro^{44 (link)} through ImageJ software (National Institutes of Health) as previous described^{43 (link)}. In brief, images of 30 cells from random view field of each group were first processed with a median filter to obtain isolated and equalized fluorescence, then individual mitochondria were analyzed for the lengths of major axes.