Permanox dishes

Electron microscopy was performed as described previously [47 ]. NRVMs were seeded on gelatin-covered 35 × 10 mm Permanox dishes (Electron Microscopy Sciences, 70,340) at a density of 2 × 10⁵ cells per plate and transfected with siRNA as described above. Six days after transfection, NRVMs were rinsed with warm (room temperature), diluted 1:1 with ddH2O, cardioplegic buffer (50 mmol/L KCl, 5% dextrose in PBS, pH 7.4) and fixed on ice in 1% paraformaldehyde/2% glutaraldehyde in 50 mM cacodylate buffer, pH 7.2, for 20 min, postfixed in reduced osmium (1% OsO4, 0.75% K3Fe(CN)6, 25 mM cacodylate buffer, pH 7.2), dehydrated using a graded series of acetone, and embedded in epoxy resin. After polymerization thin layer containing cell monolayers were detached from the bottom of the dish, cut into small fragments and glued to stubs for sectioning. Ultrathin sections were counterstained with uranyl acetate and lead salts. Images were acquired on a Hitachi 7600 electron microscope equipped with an Advanced Microscopy Techniques digital camera.