Progeny Varicella-Zoster Virus Capsids Exit the Nucleus but Never Undergo Secondary Envelopment during Autophagic Flux Inhibition by Bafilomycin A1

Girsch, James H.; Walters, Katherine; Jackson, Wallen; Grose, Charles

Transmission electron microscopy. Cell culture medium overlying a monolayer was replaced with 2.5% glutaraldehyde in 0.1 M cacodylate buffer prewarmed to 37°C (53) . The cells undergoing fixation were allowed to cool to room temperature and then remained in the fixative overnight at 4°C. After rinsing several times in 0.1 M cacodylate buffer, postfixation was carried out for 20 min with 1% osmium tetroxide solution reduced with 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer solution (54) . This second fixative was removed with several rinses of distilled water. An en bloc staining with 2.5% uranyl acetate was done for 5 min. The cells were then dehydrated using gradually increasing concentrations of ethanol to 100%. At this point, infiltration of a 1:1 mixture of Eponate 12 epoxy resin (Ted Pella) and ethanol was applied and incubated for 1 h at room temperature. This mixture was replaced with 100% resin three times at 1-h intervals and cured for an additional 48 h in a 60°C oven. Sections 80 nm thick were cut using a Leica UC-6 ultramicrotome and collected on Formvar-coated 75-mesh copper grids. Counterstaining was done with 5% uranyl acetate for 2 min and with Reynold's lead citrate for 2 min (55). Images were collected at 120 kV using a JEOL 1230 transmission electron microscope and recorded directly in digital format with a Gatan 2,000-by-2,000 charge-coupled-device camera equipped with Gatan Microscopy Suite software.