Analysis version docu software

Infected HeLa cells were washed with PBS (3×) and cooled on ice before fixation with 0.5% glutaraldehyde (Agar Scientific) in 200 mM sodium cacodylate (TAAB) for 5 min on ice, then at RT for a further 25 min. Cells were immediately washed in cacodylate buffer and Tf-HRP reacted with diaminobenzidine (DAB) in stable peroxide buffer (Metal Enhanced DAB Substrate Kit, Thermo Scientific) for 30 min at RT. The reaction was stopped by washing in sodium cacodylate before post-fixation in 1% osmium tetroxide/1.5% potassium ferrocyanide for 1 h at RT. The cells were then washed in ddH₂O, stained overnight at 4°C with 0.5% uranyl acetate, washed with ddH₂O and serially dehydrated in graded ethanol before infiltration with Epon 812 resin. Ultrathin sections (∼70 nm) of the flat-embedded cell monolayers were cut parallel to the surface of the dish, collected onto formvar-coated 50 mesh EM grids, and stained for 30 s with Reynolds' lead citrate before imaging. TEM samples were viewed by using an FEI Tecnai G² electron microscope with a Soft Imaging System Megaview III charged-coupled-device camera. Images were collected at 1376 by 1032 by 16 pixels using AnalySIS version Docu software (Olympus Soft Imaging Solutions).

MA‐NSP5‐EGFP cells were seeded at 1 × 10⁶ cells in 6 cm² wells and infected at MOI of 10. 12 hpi, cells were fixed with 0.5% glutaraldehyde in 200 mM sodium cacodylate buffer, pH 7.4, first on ice for 5 min, then at room temperature for 25 min, and washed 3 times with 200 mM sodium cacodylate buffer. Samples then were postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h at room temperature, washed with distilled water and stained in 0.5% magnesium‐uranyl acetate in water at 4°C overnight. Cells were washed with distilled water and dehydrated in a graded ethanol series starting at 70%, followed by two changes into absolute alcohol, and embedded in Epon resin. Ultrathin sections (70 nm) were cut parallel to the surface of the dish using a Leica ultramicrotome. The sections were collected onto 50 mesh formvar grids and stained with Reynold’s lead citrate for 30 s, washed with water and air‐dried. Samples were viewed with a FEI Tecnai G² electron microscope with a Soft Imaging System Megaview III CCD camera. Images were collected at 1,376 × 1,032 × 16 pixels using AnalySIS version Docu software (Olympus Soft Imaging Solutions).