Ultrastructural Analysis of Mitochondria

Cells grown on glass coverslips were fixed overnight at 4 °C in 2% glutaraldehyde in 0.1 M phosphate buffer, before being incubated for 1 h in 2% osmium tetroxide in 0.1 M phosphate buffer, dehydrated through a series of graded ethanol concentrations, and embedded in EM-bed (Electron Microscopy Sciences). The glass coverslips were removed by treatment with hydrofluoric acid. Next, 100 nm sections were cut on a Leica Ultracut EM UC7 ultramicrotome and stained with uranyl acetate and lead citrate. The grids were viewed at 80 kV in a JEOL JEM-1400 transmission electron microscope and images were captured by an AMT BioSprint digital camera. For quantitative analysis, fields were chosen at random, and acquisition and quantification were performed using FIJI (NIH) and GraphPad Prism 9.3.1 software, respectively. Morphometric analyses of the mitochondria and substructures were performed by three investigators in a randomized, double-blind manner, which included taking the images, quantifying the data, and running the statistics separately. Images were obtained at random from three independent experiments^{[29 (link)]}.