2.6. Immunofluorescence staining

For MitoTracker staining of mitochondria, fibroblast and HeLa cells were seeded at a density of 2.5 × 10³ per well (24 well plate) on glass coverslips. The MitoTracker lyophilized probe was reconstituted in anhydrous DMSO to a stock concentration of 1 mM. A working concentration of 100 nM was established by dilution in nutrient media prior to addition to cells and incubated at 37 °C in 5% CO₂ for 30 min. After incubation, cells were washed twice with phosphate buffered saline (PBS) and coverslips fixed with 4% paraformaldehyde for 15 min. Permeabilization was achieved by addition of 0.2 % Triton X-100 for 5 min. Coverslips were counterstained with DAPI and washed with PBS twice prior to mounting on microscope slides. Images were obtained using the 63x oil objective of the confocal Laser scanning microscope 710. Confocal Z-stack images of immuno-stained cells (25 cells per condition) were taken at 1 μm intervals then subjected to volumetric analysis using surface rendered 3D reconstruction by Imaris image analysis software (Imaris 7.6.1 Bitplane, Concord, USA). Morphometric analysis of reconstructed mitochondria was performed using the “ImarisSurface” in-built software tool.