2.2. Transmission Electron Microscopy

Thin-section TEM was used to characterize the ultrastructural modifications of infected cells. Cultures were processed using the previously described methodology [39]. Briefly, after the samples were fixed as described in the previous section, these were washed three times with 0.1 M sodium cacodylate buffer pH 7.2; the samples were further fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate for one hour at room temperature in the dark. The cultures were rinsed twice with the sodium cacodylate buffer and once with 0.1 sodium acetate buffer pH 4.5, block-stained with 0.5% uranyl acetate in 0.1 M sodium acetate for one hour at room temperature in the dark. Subsequently, the cells were washed three times with the 0.1 M sodium acetate and two times with MilliQ water before being left to incubate in MilliQ water overnight. The following day, cells were dehydrated with ethanol with increasing concentration (35%, 50%, 70%, 95%, and 100%) for 10 min and three times for each concentration, then cells were rinsed three times with Embed 812 resin (Electron Microscopy Science, Hatfield, PA, USA). Finally, the cells were embedded in the EMBed 812 resin and polymerized for 48 h at 55 °C. Ultrathin sections (70 nm) were obtained using an EM-UC7 ultramicrotome (Leica Inc., Karnataka, India) and transferred to 100-mesh copper grids and stained with 0.5% uranyl acetate and 0.5% lead citrate. After staining, the grids were kept in closed Petri dishes containing dry pellets of NaOH and silica. The samples were visualized in a JEM-JEOL-2100 transmission electron microscope at 200 kV using a Gatan 4K camera. The digital electron micrographs were recorded with the Digital Micrograph (Gatan Inc., Pleasanton, CA, USA) software and analyzed with ImageJ and Fiji (NIH, Bethesda, ML, USA). Each sample was done in duplicate.