Fastscan ccd camera

Primary cardiomyocytes isolated from WT and syndapin III KO mice were fixed by adding 2.5% glutaraldehyde in 0.1 M cacodylate buffer. After 1 hr, the cells were collected by centrifugation at 600 x g and washed 3x with PBS. After post-fixation in 1% osmiumtetroxide for 1 hr, dehydration in ascending ethanol series with post-staining in uranylacetate was performed. The samples were embedded in epoxy resin (Araldite) and finally sectioned ultrathin using a LKB Ultratome III (LKB). After mounting on coated copper grids and post-staining with lead citrate for 10 min, the sections were examined in a TEM (EM 902A, Zeiss) at 80 kV. Images were recorded digitally using an 1 k FastScan-CCD-camera (TVIPS camera and software). Plasma membrane sections of about 2 µm length each were analyzed for the presence or absence of caveolar membrane profiles. Data were expressed as caveolae/length of membrane stretch. Images were processed using Adobe Photoshop software.

Carbon-coated grids (400 meshes) (Quantifoil, Großlöbichau, Germany) were hydrophilized by glow discharge at low pressure in air. Subsequently, 20 μl a solution of purified Emp_FL (3.1nmol) or Emp₂₊₃ (2.4nmol), was adsorbed onto the hydrophilic grids for 1 min. The grids were washed twice with drops of distilled water and were stained with a drop of 2% uranyl acetate in distilled water. The samples were analysed using a Zeiss EM902A electron microscope (Carl Zeiss AG, Oberkochen, Germany) operated at an acceleration voltage of 80 kV. Images were recorded with a FastScan-CCD camera at 1024 × 1024 pixels (TVIPS, Munich, Germany).

Sample preparation was done as described in Ref. [33 (link)]. The freeze‐fracture replicas were imaged in a Zeiss EM902A transmission electron microscope (Carl Zeiss). Images were recorded with a 1k (1024 × 1024) FastScan‐CCD‐camera (CCD‐camera and the acquisition software emmanu4 v 4.00.9.17, TVIPS, Munich, Germany).