Cells

The cultures were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde. The cells were then rinsed with 0.15 M sodium cacodylate (pH 7.2–7.4) for 10 min and incubated in fresh 1% osmium tetraoxide in 0.1 M sodium cacodylate for 1 h at RT. After incubation, the sodium cacodylate was rinsed away to dehydrate the dishes with 70% ethanol for 30 min, 95% ethanol for 30 min and > 99% ethanol for 1 h. A thin plastic layer (Agar 100 resin kit, Agar Scientific Ltd) was added to the dishes and incubated for 1 h. The plastic was poured off and a new plastic layer was added onto the dishes for incubation overnight in a desiccator. Next, the plastic was heated to enable its removal after which a new thicker plastic layer was added before another incubation for 1 h in a desiccator. Cells were covered with 3-mm plastic and polymerized in the oven at 60 °C for 48 h. Embedded cells were sectioned by using a Leica ultracut UTC ultramicrotome (Rowaco AB) and visualized with a Tecnai G2 transmission electron microscope (FEI company) with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).