Leica em stain

For ultrastructural analysis, conventional, and microwave assisted fixation, substitution, and resin embedding of isolated cells was performed as described in Supplementary Table S1. Ultrathin sections of approximately 70 nm were cut with a diamond knife, transferred onto TEM grids and contrasted in a LEICA EM STAIN (Leica Microsystems, Vienna, Austria) with uranyl acetate and Reynolds’ lead citrate. Ultrastructural analysis was performed using a Tecnai Sphera G2 TEM (FEI, Eindhoven, Netherlands) operated at 120 kV.

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)].

Aliquots of freshly isolated mitochondria-enriched fractions were fixed with 2.5% glutaraldehyde (v/v) in 0.1 M Sørensen phosphate buffer (0.1 M Na₂HPO₄, 0.1 M NaH₂PO₄, pH 7.2). Isolated mitochondria were centrifuged at 14,000× g for 10 min. The resulting pellets and BAT were washed in PB and postfixed in 2% osmium tetroxide in the same buffer, then routinely dehydrated using increasing concentrations of ethanol and embedded in Araldite. Ultra-thin sections of isolated mitochondria and BAT were obtained using a Leica UC6 ultramicrotome (Leica Microsystems), mounted on copper grids, and contrasted in uranyl acetate and lead citrate using Leica EM STAIN (Leica Microsystems). Sections were examined on a Philips CM12 transmission electron microscope (Philips/FEI, Eindhoven, The Netherlands) operated at 60 kV and equipped with the digital camera SIS MegaView III (Olympus Soft Imaging Solutions, Münster, Germany). The obtained electron micrographs (pixel size: 8 × 8 nm²) were used for confirmation of mitochondrial isolation.