Megaview g3

Flies were decapitated and the heads were perfused with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and 5 mM CaCl₂ overnight. Brains were then treated with 1% osmium tetroxide for 3 h at 4°C and embedded in Araldite® CY212 resin. Thin sections were stained with toluidine blue and the brain visualized with the light microscope. Ultrathin sections (70 µm) were stained with lead citrate and uranyl acetate and digital images taken on a Philips CM10 TEM with MegaView G3 digital imaging system with Radius Software (Olympus). Mitochondrial morphology was measured blind to genotype.

Fibroblast cell lines were established from skin biopsies of proband AII-2 and a healthy age- and sex-matched control using standard methods. Cells were grown in high glucose Dulbecco's modified Eagle’s medium, supplemented with 10% fetal bovine serum, penicillin/streptomycin, and 0.05 mg/mL uridine. For electron microscopy (EM), fibroblasts were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer and 5 mM CaCl₂ overnight, then treated with 1% osmium tetroxide for 3 hours at 4°C and embedded in Araldite CY-212 resin. Ultrathin sections (70 μm) were stained with lead citrate and uranyl acetate. Images were taken on a Philips CM10 transmission electron microscope fitted with a MegaView G3 camera and RADIUS Software (Olympus). Mitochondria morphology in fibroblasts was measured blind to disease status.