Araldite 502 embed 812 kit

Cells grown on glass coverslips were fixed for 30 min by incubation with pre-warmed 2.5% glutaraldehyde/2% sucrose in 50 mM sodium cacodylate buffer (CaCo)(50 mM cacodylate, 50 mM KCl, 2.6 mM CaCl₂, 2.6 mM MgCl₂, pH-7.4). After 3 washes with 50 mM CaCo buffer, cells were incubated with 2% osmium tetroxide/50 mM CaCo for 40 min on ice, washed with water 3 times and treated with 0.5% uranyl acetate for 30 min. After 30 min rinsing with water, cells were progressively dehydrated with increasing concentrations of ethanol (40% to 100%) and finally infiltrated in a polymerizing Epon/araldite resin (Araldite 502/Embed 812 kit; Electron Microscopy Sciences) for 72 h at 60°C. Embedded cells were sectioned into 70-nm-thick slices by using an Ultracut UCT microtome (Leica) and a diamond knife (Diatome). After counterstaining with 3% uranyl acetate in 70% methanol for 5 min and 2% lead citrate in water for 2 min, cells were examined either with an EM-10 transmission electron microscope (Zeiss) with a built-in MegaView camera (Olympus) or with a JEOL JEM-1400 transmission electron microscope (Jeol Ltd., Tokyo, Japan). Quantification of virion number, size, and distribution was done manually or using macros developed for the Fiji software package.