Lx 112

Fixed samples were osmicated 1–2 h with 1.5% (w/v) reduced OsO₄ in 100 mm cacodylate, pH 7.4, washed several times with distilled water, and then block stained overnight at 4 °C in 0.5% (w/v) aqueous uranyl acetate (Electron Microscopy Sciences, Hatfield, PA). Tissues were dehydrated in a graded series of ethanol, embedded in the epoxy resin LX-112 (Electron Microscopy Sciences), and sections (pale gold in color) were cut with a Diatome diamond knife (Electron Microscopy Sciences). Sections were counterstained with uranyl acetate and lead citrate and viewed on a JEOL 1011 transmission EM with a side mount AMT 2K digital camera (Advanced Microscopy Techniques, Danvers, MA). Images were imported into Photoshop CC (Adobe, San Jose, CA), adjusted for brightness and contrast, and then assembled in Adobe Illustrator CC. All of the EM studies were performed using an n = 3 for experimental and control. While our study does not permit the quantification of the frequency of each phenotype (mitobodies, and junctional defects), these abnormalities were readily observed in every mutant analyzed. None of these features were observed in any of the controls. The degree to which each phenotype represents the displayed image in the figures is described below for the specific image.