Transmission electron microscopy

SC-derived primary myoblasts isolated from Pax7^CreERT2;Pnpla2^flox/flox mice were treated with 4-OHT (0.4 μM, Calbiochem) to induce genetic deletion. Cultured primary myoblasts and isolated tibialis anterior muscles were fixed in a fixative buffer (2.5% glutaraldehyde, 1.5% paraformaldehyde in 0.1M sodium cacodylate buffer) and scraped for collection. Samples were rinsed in deionized water followed by fixation in 2% osmium tetroxide for 1 h. Then, the samples were washed in deionized wat water followed by fixation in 1% uranyl acetate for 15 minutes. After washing with deionized wat water, the samples were dehydrated with series of graded ethanol followed by dehydration in acetonitrile and then were embedded (EMbed 812: DDSA: NMA 5:4:2; 0.22 DMP-30). Ultrathin sections (longitudinal sections) were cut at 70 nm and stained with uranyl acetate and lead citrate. Stained sections were examined under Tecnai T12 transmission electron microscope attached with a Gatan imaging system under 6,000× and 260,00× magnifications.