Tem 912

Ultrathin sections (60 nm) of embedded cells were cut using an ultramicrotome (Reichert Ultracut S, Leica, Germany) with a diamond knife (Diatome, Switzerland) and mounted on 300-mesh copper grids (Plano, Germany). Sections were examined, without further staining, using a Zeiss transmission electron microscope 912 (TEM-912, Carl Zeiss, Germany) operating at 80 kV and equipped with a digital camera (Proscan 2 K Slow-Scan CCD-Camera, Carl Zeiss, Germany). Digital image acquisition was performed using the iTEM software (Olympus GmbH, Germany)^{2 (link),18 (link)}.

All postembedding steps except for the incubation with primary antibodies were performed at room temperature. For single and double immunolabeling with SNAP47 antibody, sections were first incubated two times for 5 min in 0.1 M PBXT (PBS, 0.001% Triton X-100, 0.001% Tween 20, pH = 7.4), followed by 90 min incubation in PBXT supplemented with 2% bovine serum albumin (BSA, Sigma-Aldrich, Darmstadt, Germany) and 5% normal goat serum (NGS; PAN Biotech) at room temperature. The sections were next incubated with primary antibodies diluted in the same buffer overnight at 4°C in a humid chamber. After rinsing several times with PBXT, the binding of primary antibodies was visualized by incubating with goat anti-rabbit or goat anti-guinea pig secondary antibodies conjugated to either 5 or 10 nm gold particles (British BioCell, International, Wetzlar, Germany) in PBXT supplemented with 0.5% acetylated BSA (Aurion, Wageningen, Netherlands), for 90 min in a humid chamber. Grids were rinsed several times in PBXT, PBS, and finally in water. Ultrathin sections were finally stained with 2% aqueous uranyl acetate (Merck, Darmstadt, Germany) for 2 min, and with lead citrate for 30 s. Sections were examined using a Zeiss TEM-912 equipped with a digital camera (Proscan 2K Slow-Scan CCD-Camera, Zeiss, Oberkochen, Germany). For negative controls, primary antibodies were omitted.