Epon resin

U2OS cells were grown to 70% confluency in 35mm glass bottom Grid-500 μ-Dish (Ibidi, Cat.No. 81168) and infected with Alexa594 fluorescently labeled WT or M1 viruses (250 physical particles per cell) at 37°C for 30 min. At 30 min post infection cells were fixed with 2.5% glutaraldehyde (GA) in PBS overnight at 4°C. Subsequently, the cells were washed with PBS, postfixed for 30 minutes with 1% OsO4 in PBS, washed with ddH2O, and stained with 1% uranyl acetate in water. The samples were gradually dehydrated with ethanol and embedded in Epon resin (Carl Roth, Germany) for sectioning. Ultrathin 50 nm sections were prepared using Ultracut Microtome (Leica Microsystems, Germany). The sections were poststained with 2% uranyl acetate. Electron micrographs were obtained with a 2K wide angle CCD camera (Veleta, Olympus Soft Imaging Solutions GmbH, Münster, Germany) attached to a FEI Tecnai G 20 Twin transmission electron microscope (FEI, Eindhoven, The Netherlands) at 80kv.

Matrigel‐coated beads covered with cells were harvested, rinsed with PBS and fixed with 2.5% glutaraldehyde (Serva, Heidelberg, Germany) in 0.1 M sodium cacodylate buffer (Serva, Heidelberg, Germany) for 30 min at RT and stored at 4°C. The samples were postfixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, USA) and 0.8% potassium ferrocyanide II (Roth, Karlsruhe, Germany) in 0.1 M cacodylate buffer for 1.5 h and embedded in agarose overnight. After cutting the agarose in smaller blocks, the samples were dehydrated in a graded ethanol series and transferred to Epon resin (Roth, Karlsruhe, Germany). Finally, ultrathin sections of the samples (70nm) were stained with uranyl acetate, and lead citrate. The examination was carried out with a Zeiss EM 906 electron microscope at 80kV acceleration voltage (Carl Zeiss, Oberkochen, Germany).