Morgagni 268d

Glutaraldehyde-fixed fragments of cortical kidney tissue were washed repeatedly in cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated through ascending grades of alcohol, and embedded in Epon resin. Semithin sections were stained with toluidine blue in borax and examined using light microscopy. Thin sections (100–120 nm) were stained with uranyl acetate for morphologic analysis using a Philips Morgagni transmission electron microscope (Morgagni 268D; Philips, Brno, Czech Republic). The mitochondrial numerical density was estimated on 30 digitised TEM pictures at × 7100 for each sample and expressed as number of mitochondria per unit area (N_A, n/µm²). Briefly, the mitochondrial profile area density (NA) was estimated using the ratio between the number of mitochondria and the cellular area in the image, using interactive image editing software (ImageJ; National Institutes of Health, http://rsbweb.nih.gov/ij/). Mean mitochondrial volume at W8 was measured using digitized TEM images as previously described^{28 (link)}. To evaluate the extent of mitochondrial alterations, the number of mitochondria with invaginations per total number of mitochondria observed was quantified in n = 752 mitochondria for control and n = 615 for Sirt3^−/− mice, using interactive image editing software (ImageJ).

Fragments of kidney tissue were processed as described54 (link). Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a transmission electron microscope (Morgagni 268D, Philips, Brno, Czech Republic).