Epon and araldite resin mixture

Cells were pelleted, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 24 h at 4°C and washed with 0.1 M phosphate buffer. 1% melted agar (Sigma-Aldrich) was added to the pelleted cells and placed at −20°C for 5 min. The solidified cell pellet–agar block was post-fixed with 1% osmium tetroxide, dehydrated in a graded ethanol series, cleared in propylene oxide and embedded in an epon and araldite resin mixture (Agar Scientific). Ultrathin (80 nm) sections were prepared on a Reichert UCT ultramicrotome (Leica). Sections with a silver-gold interference colour were mounted on 200 mesh copper grids (Agar Scientific) and contrasted with uranyl acetate and lead citrate. Images were obtained on a JEM 1010 transmission electron microscope (JEOL) equipped with a Mega View III digital camera (Olympus).

C2C12 myoblasts cultured in 35-mm cell culture dishes were treated with either TM or DMSO for 16 h and fixed in situ in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 1 h at room temperature. The cells where then detached, pelleted and washed three times with 0.1 M phosphate buffer (pH 7.2). 1% melted agar (Sigma-Aldrich) was added to the pelleted cells, and they were then placed at −20°C for 5 min. The solidified cell pellet–agar block was post-fixed with 1% osmium tetroxide (BDH Chemicals) for 1 h at room temperature, dehydrated through a graded ethanol series, cleared in propylene oxide and embedded in an epon and araldite resin mixture (Agar Scientific). The resin was polymerized at 60°C overnight. Ultrathin sections of 80–100 nm thickness were prepared on a Reichert-Jung UCT ultramicrotome (Leica), mounted on 200-mesh copper grids (Agar Scientific) and stained with uranyl acetate and lead citrate. Sections were observed on a JEM 1010 transmission electron microscope (JEOL) equipped with a Mega View III digital camera (Olympus).