Analysis of mitochondrial morphology by transmission electron microscopy

Cells were fixed for 1 hour at 65°C in 2% paraformaldehyde and 2.5% glutaraldehyde (Polysciences, Warrington, Pennsylvania, USA) in 100 mM sodium cacodylate buffer (pH 7.2). Samples were washed in cacodylate buffer and then postfixed for 1 hour in 1% osmium tetroxide (Polysciences). Samples were then extensively rinsed in distilled H₂O before undergoing en bloc staining for 1 hour with 1% aqueous uranyl acetate (Ted Pella). After several rinses in distilled H₂O, the samples were dehydrated in a graded series of ethanol and then embedded in Eponate 12 resin (Ted Pella). Sections (95 nm in thickness) were cut with an Ultracut UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1400 transmission electron microscope (JEOL USA) equipped with an XR611 high-resolution, 11-megapixel mid-mount charge-coupled device camera (Advanced Microscopy Techniques, Woburn, Massachusetts, USA).