2k 2k ccd camera

Samples were prepared for EM as previously described [13 (link)]. Briefly, cells were fixed in the fixative media (1% glutaraldehyde, 0.2% paraformaldehyde, and 40 mM potassium phosphate, pH 7.0) for 10 min at RT followed by incubation on ice for 50 min in fresh fixative media. Cells were washed twice with 0.9% NaCl and once with water. Cells were incubated with 2% solution of KMnO₄ for 5 min at RT, centrifuged and again resuspended with fresh solution of 2% KMnO₄ for 45 min at RT for en-bloc staining. Subsequently cells were dehydrated using graded series of ethanol, embedded stepwise using Spurr’s low viscosity resin (EMS, Hatfield, PA, USA), and polymerized at 65°C for 48 hr. Semi and ultrathin sections were produced with a diamond knife (Diatome, Biel, Switzerland) on an ultra-microtome (UC7, Leica-Microsystems), collected on 200 mesh copper grids (EMS, Hatfield, PA, USA), post stained with uranyl acetate and lead citrate, and visualized with a Tecnai T12 TEM (FEI), operating at 120 kV. Pictures were recorded on a below mounted Gatan 2k × 2k CCD camera.