Em 1010 transmission electron microscope

The specimens were fixed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer (0.1 mol L -1 pH 7.4) during ca. 12 h at 4 °C and constant darkness. Then, the fixed specimens were washed with the same cacodylate buffer and post-fixed in 1% osmium tetroxide solution in cacodylate buffer during 90 minutes at 4 °C. The post-fixed specimens were washed again with cacodylate buffer, double-distilled water, and dehydrated in a graded series of acetone (30%, 50% and 70% realizing one time each step, followed by 90%, 95% and 100% realizing three times each step, in all cases were used 15 min step). The post-fixed and clean specimens were embedded in Spurr's resin. The semi-thin slices were obtained using a Leica UCT ultramicrotome (Leica, Wetzlar, Germany) and stained with Toluidine Blue. The ultrathin slices were obtained using the same ultramicrotome and stained with uranyl acetate and lead citrate. Observations were made with a Jeol EM-1010 transmission electron microscope (tungsten filament, 80 kV). Post-fixing procedures and observations were realized at Centres Científics i Tecnològics de la Universitat de Barcelona (CCiTUB; Hospital Clinic, Barcelona, Spain).