Tecani12 biotwin

Cells were initially fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and further processed for cryo immunogold labelling as previously described^{91 (link)}. From the resulting frozen sample blocks, 50 nm ultrathin sections were cut at −110 °C. The sections were retrieved in a mixture of sucrose/methylcellulose and transferred on formvar coated copper grids (200-mesh, hexagonal). Single immunogold labelling was performed against HA-tagged Lima1 with an antibody recognizing HA-tag (mouse, clone 16.B12, Biolegend), detected by a bridging antibody, which was recognized by 10 nm protein A gold conjugate (CMC, Utrecht, Netherlands).
For double immunogold labelling antibodies against actin (mouse, 4F7, kind gift from Professor Brigitte Jockusch and Sabine Buchmeier, Antibody Facility TU-Braunschweig) and Lima1 (rabbit, A300-102A, Bethyl Laboratories, Inc.) were used stepwise, detected by 15 nm and 10 nm protein A gold, respectively. The samples were analyzed at 80 kV using a transmission electron microscope (Tecani12-Biotwin, Thermo Fisher Scientific). Representative images were exposed on ditabis imaging plates (Ditabis, Pforzheim, Germany).

HUVEC were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M PHEM buffer, pH 7.2. Samples were processed further for cryosectioning and immuno gold labelling as described.^[73 (link)
^] For double immunogold labelling of ultrathin cryosections the GDPD5 protein was marked with the rabbit antibody, diluted 1:20 in the first step, labelled by 10 nm protein A gold (CMC, Utrecht, the Netherlands). After stabilizing the components with 1% glutaraldehyde the second labelling step was directed against VWF, using the rabbit antibody diluted 1:100 and subsequently decorated with 15 nm protein A gold. Sections were analyzed at a transmission electron microscope, operated at 80 kV (Tecani12‐Biotwin, Thermo Fisher Scientific). Representative images were acquired on ditabis imaging plates (Ditabis, Pforzheim, Germany) and on a 2 K CCD camera (Veleta, EMSIS, Münster, Germany).