Single computed slice through a dual axis tilt electron tomographic reconstruction of double membrane vesicles in a freeze-substituted VeroE6 cultured cell infected with the SARS-CoV. This image has been downsampled from the raw data image which can be accessed using the link provided to the Cell Centered Database. For more information, see: Knoops et al. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol. 2008 6(9):e226. PMID: 18798692
For ultrastructural morphological investigations, SARS-CoV-infected Vero E6 cells were pre-fixed (for biosafety reasons) overnight with 3% paraformaldehyde in 0.1M PHEM buffer (60 mM piperazide-1,4-bis[2-ethanesulfonic acid], 25 mM HEPES, 2 mM MgCl2, 10 mM EGTA) at various time points after infection. For cryo-fixation, cell monolayers adhered to Thermanox coverslips (Nunc, Denmark) were plunged into liquid ethane. Freeze substitution was performed at -90°C in an automated freeze-substitution system (Leica, Austria) using an FS medium consisting of 90% acetone and 10% water, containing 1% osmium tetroxide and 0.5 % uranyl acetate. After washing with pure acetone at RT, the samples were embedded in epoxy LX-12 resin. Thin sections were collected on 100 Mesh grids, 1.25% pioloform support film contrasted with uranyl acetate and lead hydroxide and subsequently viewed at 80 kV with a Philips CM-10 transmission electron microscope (Philips, The Netherlands).
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