Biotwin cm120 transmissionelectron microscope

Samples before and after TDP treatment were stored in 20% dextran-500 (Sigma Aldrich, St. Louis, MO, USA) phosphate buffered saline (PBS) (Gibco) overnight to minimize any swelling effects before fixation. The fixative contained 3% paraformaldehyde, 1.5% glutaraldehyde, 5 mM MgCl₂, 5 mM CaCl₂, 2.5% sucrose, and 0.1% tannic acid in 0.1 M sodium cacodylate buffer, at pH 7.2. Samples were fixed overnight at 4 °C, then post-fixed in 1% osmium tetroxide, followed by staining with 2% aqueous uranyl acetate and dehydrated in increasing concentrations of ethanol (from 30% to 100%). Thin sections (60–90 nm) were cut and stained with lead and uranyl acetate. Sections were observed with Philips/FEI BioTwin CM120 Transmission Electron Microscope at 80 kV.

Cultures were fixed and processed for electron microscopy (Clayton et al. 2008). Cultures were either incubated with DMSO (vehicle), BAPTA‐AM or EGTA‐AM (both 100 μM) in incubation medium for 30 min before stimulation with a train of 800 action potentials in the presence of 10 mg/mL horse radish peroxidase (HRP). TnTx‐treated cultures were subjected to the same stimulus and loading, but without the 30 min incubation period. Cultures were then immediately fixed in 2% glutaraldehyde in phosphate‐buffered saline. After washing with 100 mM Tris (pH 7.4), cultures were exposed to 0.1% diaminobenzidine and 0.2% H₂O₂ in 100 mM Tris until colour developed. Cultures were then washed with 100 mM Tris, stained with 1% osmium tetroxide for 30 min and then dehydrated and embedded in TAAB LEMIX resin (TAAB Laboratories Equipment, Aldermaston, UK). Ultrathin sections were cut and mounted on grids, stained with uranyl acetate and lead citrate and viewed using a Philips BioTWIN CM120 transmission electron microscope Intracellular structures that were < 100 nm in diameter were arbitrarily designated to be SVs, whereas larger structures were considered endosomes.