Tungstophosphoric acid

Cells were subjected to velocity sedimentation centrifugation, and three intermediate fractions were pooled and dialyzed for 18 h against phosphate-buffered saline at 4 °C. Dialyzed samples were then settled onto carbon-coated grids for 2 to 3 min, stained with 1% uranyl acetate for 30 s, and visualized with a Zeiss EM 10C/CR transmission electron microscope (Stockholm, Sweden). Studies were performed using a Philips CM100 electron microscope (Philips, Eindhoven, The Netherlands). After fixation in 1.25% glutaraldehyde, virus particles were pelleted on carbon/Formvar-coated 400-mesh copper grids (GilderGrids, Lincolnshire, UK). Briefly, 150 μL of virus suspension was centrifuged for 10 min in an Eppendorf 5417C centrifuge (Hamburg, Germany) with a swing-out rotor at a maximum force of 12,000× g. The grids were placed on the flat bottom of the outer container of Sarstedt microvette CB 300 tubes (Nümbrecht-Rommelsdorf, Germany). Grids were stained by 2% tungstophosphoric acid (Merck, Darmstadt, Germany) at pH 6.

IPSC-CM were fixed with 4% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.3 and prepared for electron microscopy essentially as described [47 (link)]. In brief, the adherent cultures were postfixed on the culture dish with 1% OsO4 (Science Services, Munich, Germany) in 0.1 M phosphate buffer and embedded in Epon (Serva, Heidelberg, Germany) after dehydration with ethanol and en bloc staining with 1.5% uranyl acetate (Merck, Darmstadt, Germany) and 1.5% tungstophosphoric acid (Merck, Darmstadt, Germany) in 70% ethanol. Ultrathin sections of cultured cells were cut parallel to the substrate using an UC7 Ultramicrotome (Leica, Vienna, Austria) and stained with UranyLess® (Science Services, Munich, Germany). Sections were analyzed with an LEO EM912 Omega (Zeiss, Oberkochen, Germany) and digital micrographs were obtained with an on-axis 2048 × 2048-CCD camera (TRS, Moorenweis, Germany).