Lab tek chamber slides of 2 wells

Cells were seeded at 2000 cells/cm² in Lab-Tek chamber slides of 2 wells (Nalge Nunc International, Naperville, IL) and were fixed in 3% glutaraldehyde for 1 hour at 37°C. Cells were postfixed in 2% OsO4 for 1 hour at room temperature and stained in 1% uranyl acetate in the dark for 2 h at 4°C. Finally, cells were rinsed in distilled water, dehydrated in ethanol, and infiltrated overnight in Durcupan resin (Fluka, Sigma-Aldrich, St. Louis, USA). Following polymerization, embedded cultures were detached from the chamber slide and glued to araldite blocks. Serial semithin sections (1.5 μm) were cut with an Ultracut UC-6 (Leica, Heidelberg, Germany) and mounted onto slides and stained with 1% toluidine blue. Selected semithin sections were glued with Super Glue-3, Loctite (Henkel, Düsseldorf, Germany), to araldite blocks and detached from the glass slide by repeated freezing (in liquid nitrogen) and thawing. Ultrathin sections (0.06–0.08 μm) were prepared with the Ultracut and stained with lead citrate. Finally, photomicrographs were obtained under a transmission electron microscope FEI Tecnai G2 Spirit (FEI Europe, Eindhoven, Netherlands) using a digital camera Morada (Olympus Soft Image Solutions GmbH, Münster, Germany).