Sodium cacodylate

Caco-2 cells were seeded at 7.5 × 10⁴ cells/cm² on transwell inserts of 24 well plates (polyester membrane, pore size 0.4 µm; Corning Life Sciences, Amsterdam, Netherlands) and cultured until the cells formed a confluent monolayer and further 7, 14 or 21 days. For transmission electron microscopical analysis, the cells were treated according to the protocol of Bye et al. [32 (link)]. They were washed once with 0.1 M sodium cacodylate (pH 7.4) and fixed with a solution containing osmium tetroxide and glutaraldehyde as fixatives (0.5% osmium tetroxide (Polysciences Europe GmbH, Eppelheim, Germany), 2.5% glutaraldehyde, 10 mM potassium hexacyanoferrate(II) (both from Sigma-Aldrich/Fluka, Taufkirchen, Germany), 0.05 M sodium cacodylate (Carl Roth, Karlsruhe, Germany)). After fixation the cells were washed once again with sodium cacodylate, dehydrated over a serial dilution with ethanol and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a Zeiss EM910 electron microscope; images were taken at 2,500× and 12,500× magnification.

For transmission electron microscopy (TEM) study, cortical neurons were plated on Aclar embedding film (Electron Microscopy Sciences). At 10 DIV, cells were fixed with fixative buffer (2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4) for 1 h at 4 °C. After washing with washing buffer (0.1 M sodium cacodylate (Sigma), 4% sucrose, 0.05% CaCl₂) for 5 min, cells were post-fixed with 1% OsO₄ (Electron Microscopy Sciences) in 0.1 M sodium cacodylate for 1 h at 4 °C, washed with cold ddH₂O, for 5 min three times, and incubated in 1% uranyl acid (Polysciences) for 1 h at 4 °C. The samples were then dehydrated with graded ethanol solutions at RT for 7 min for each step: 50% once, 70% once, 90% once and 100% three times. Then, cells were filtrated with a series of solutions as follows: (1) EtOH:Spurr's Resin (Low Viscosity Embedding Media Spurr's, Kit Electron Microscopy Sciences, Hatfield, PA)=1:1 for 30 min; (2) EtOH:Spurr's Resin=1:2 for 40 min; (3) pure Spurr's Resin for 1 h. Cells were then polymerized at 70 °C for 20 h. Ultrathin sections were sectioned with a diamond knife (DiATOME) and stained with 4% uranyl acid for 5 min followed by lead citrate stain for 8 min. After washing with ddH₂O, cells were examined by TEM (Tecnai G2 Spirit TWIN, FEI Company) with a Gatan CCD Camera (794.10.BP2 MultiScanTM) and acquisition software DigitalMicrograph (Gatan).