PMC:7321036 / 88379-90182 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7321036","sourcedb":"PMC","sourceid":"7321036","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7321036","text":"Electron microscopy\nVero E6 cells were seeded overnight and then infected for 24 hours with SARS-CoV-2 isolate nCoV-WA1-2020 on silicon chips for scanning electron microscopy or Thermanox for transmission electron microscopy. For scanning electron microscopy (SEM), cells were fixed with Karnovsky’s formulation of 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer, and then post-fixed with 1.0% osmium tetroxide/0.8% potassium ferrocyanide in 0.1 M sodium cacodylate buffer washed with 0.1 M sodium cacodylate buffer then stained with 1% tannic acid in dH2O. After additional buffer washes, the samples were further osmicated with 2% osmium tetroxide in 0.1M sodium cacodylate, then washed with dH2O. Specimens were dehydrated with a graded ethanol series from 50%, 75%, 100% x 3 for 5 minutes each, critical point dried under CO2 in a Bal-Tec model CPD 030 Drier (Balzers, Liechtenstein), mounted with double sided carbon tape on aluminum specimen mounts (Ted Pella), and sputter coated with 35 Å of iridium in a Quorum EMS300T D sputter coater (Electron Microscopy Sciences, Hatfield, PA) prior to viewing at 5 kV in a Hitachi SU-8000 field emission scanning electron microscope (Hitachi, Tokyo, Japan). For transmission electron microscopy (TEM), specimens were fixed as described above for scanning electron microscopy and additionally stained overnight with 1% uranyl acetate at 4°C after the second osmium staining and then dehydrated with the same graded ethanol series, and embedded in Spurr’s resin. Thin sections were cut with a Leica UC7 ultramicrotome (Buffalo Grove, IL) prior to viewing at 120 kV on a FEI BT Tecnai transmission electron microscope (Thermofisher/FEI, Hillsboro, OR). Digital images were acquired with a Gatan Rio camera (Gatan, Pleasanton, 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