Figure 3.

Labeling EM techniques applied to the study of the coronavirus ROs. (A) Immunoelectron microscopy (IEM) detection of dsRNA inside the DMVs induced by SARS-CoV in Vero E6 cells (adapted from [15]). On-section immunogold labeling was performed on sections from plunge-frozen freeze-susbstituted cells using a primary antibody (Ab), a secondary antibody, and protein A conjugated with 10 nm gold. (B) EM autoradiography detection of active viral RNA synthesis showing it is associated to coronaviral DMVs. Tritiated uridine was provided to live Huh7 cells infected with MERS-CoV, so that the radioactive label could be incorporated into newly-synthesized viral RNA. After 30 min, the cells were chemically fixed, prepared for EM, sectioned, and covered by a thin layer of photographic emulsion. In EM autoradiography, the radioactive disintegrations that arise from the sample in random directions create defects in the emulsion that give rise to electron-dense silver grains upon development. The image shows the high density of label in areas containing MERS-CoV-induced DMV (adapted from [39]). Scale bars, 500 nm.