Fastscan f114

A drop of diluted (1:1 in Tris buffer) sample was placed on a holey copper grid (Quantifoil R 1.2/1.3, 400 mesh) and rapidly frozen in liquid ethane (about −180°C). A cryo-transfer unit (Gatan 626, Gatan Inc., U.S.) was used to transfer the frozen specimen into the pre-cooled cryo-transmission electron microscope (CM 120, Philipps, Netherlands). The specimen was viewed under low dose conditions (120 kV), and images were recorded with a CCD camera (FastScan F114, TVIPS GmbH, Germany).

Freeze fracturing of liposomes incubated with proteins (2 µM) was done as described (Beetz et al., 2013 (link)). Cryo-TEM of liposomes coated with GST-syndapin III and syndapin III and of control incubations was performed on holey carbon film-covered copper grids. Liquid ethane-frozen samples (~−180°C) were transferred into a pre-cooled cryo-transmission electron microscope operated at 120 kV (Philips CM 120) using a cryo-transfer unit (Gatan 626-DH). Images were recorded with a 1K CCD Camera (FastScan F114, TVIPS).
Freeze-fracture replica were viewed using a Zeiss EM 902A transmission electron microscope run at 80 kV (Zeiss). Images were recorded digitally using an 1 k FastScan-CCD-camera (TVIPS camera and software).
Tubule diameters were analyzed using ImageJ. For diameter distribution analyses, measured diameters were grouped in 5 nm-step categories (0–5 nm,>5 to 10 nm,>10 to 15 nm and so on).

The purified RyR2 sample was diluted with a buffer containing 0.2 M NaCl, 20 mM MOPS-Na (pH 7.4), 2 mM dithiothreitol, and 0.015% (w/v) Tween-20, and then applied to pre-hydrophilized carbon-coated EM grids (400 mesh hexagonal copper grids, Stork Veco BV, Netherlands), negatively stained with 1.4% (w/v) uranyl acetate solution, and observed at ×40,000 magnification using a transmission electron microscope (H7500; Hitachi High-Technologies, Tokyo, Japan) operating at 80 kV. Micrographs were taken at ×40,000 using a 1024 × 1024 pixel CCD camera (Fast Scan-F114; TVIPS, Gauting, Germany).