Ultrastructural Analysis of Ventricular Mitochondria

Ultra-thin sections (80 nm thick) of ventricular tissue were obtained using a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany), mounted on copper grids and contrasted in uranyl acetate and lead citrate using Leica EM STAIN (Leica Microsystems, Wetzlar, Germany). Grids were examined on a Philips CM12 transmission electron microscope (Philips/FEI, Eindhoven, The Netherlands) equipped with the digital camera SIS MegaView III (Olympus Soft Imaging Solutions, Munster, Germany). The obtained electron photomicrographs were used for both ultrastructural and morphometric analysis of mitochondria. Mitochondrial mean diameter was calculated as the average diameter (obtained by combining diameter 1 and diameter 2 measures) for each mitochondrion, using the Image J software (Image Processing and Analyses in Java, NIH, Bethesda, MD, USA) and then mitochondria were divided in appropriate classes in dependence of their diameter. 200 SSM and 200 IFM mitochondria were randomly observed for each experimental group in almost 3 mice/group, at a final magnification of 13,000×. We excluded from the quantitative analysis perinuclear mitochondria because they are organized as clusters that make difficult to correctly analyze a single unit [40 (link)].