Tecnai transmission electron microscope

For transmission electron microscopy, 3 Mfn1^−/− and 3 WT female mice were deeply anesthetized 44 h after PMSG injection and perfused with 4% paraformaldehyde/PBS. Both ovaries were removed and fixed overnight at 4 °C with the fixative solution (paraformaldehyde 2%, glutaraldehyde 2.5% in cacodylate buffer 0.1 M, pH 7.4). After ovaries were rinsed in the same buffer twice, they were postfixed in 1% OsO4 in 0.1 M cacodylate buffer at room temperature for 60 min. Specimens were stained en bloc with 2% aqueous uranyl acetate for 30 min, dehydrated in a graded series of ethanol to 100% and embedded in Poly/bed 812 resin. Blocks were polymerized in a 60 °C oven for 24 h. Thin sections (60 nm) were cut by a Leica ultramicrotome and post-stained with 2% uranyl acetate and lead citrate. Cell sections were examined with a FEI Tecnai transmission electron microscope and digital images were recorded with an Olympus Morada CCD camera and iTEM imaging software. Follicle-enclosed oocytes were imaged at ×11500 magnification. Images were taken covering the entire follicle. ImageJ software was used to analyze the mitochondria morphology. Mitochondria area, cytoplasmic area, total mitochondria number, number of cristae, mitochondria length and width were measured. Mitochondrial coverage area was calculated by dividing the total area of mitochondria to the total area of the cytoplasm.

Twelve-month-old 5xFAD mice were perfused with 4% PFA and the brain tissues were sectioned into 50-μm-thick slices using a vibratome (VT1000S, Leica). The slices were refixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h, then post-fixed in 1% OsO₄ in the same buffer at room temperature for 1 h. After en bloc staining with 2% aqueous uranyl acetate for 30 min, tissues were dehydrated in a graded series of ethanol to 100%, followed by propylene oxide and finally embedded in EMBed 812 resin. Tissue blocks were polymerized overnight in an oven at 60 °C. Thin sections (60 nm) were cut by a Leica ultramicrotome (UC7) and post-stained with 2% uranyl acetate and lead citrate. Sample grids were examined on the FEI Tecnai transmission electron microscope with an accelerating voltage of 80 kV, and digital electron micrographs were recorded with an Olympus Morada CCD camera and iTEM imaging software.