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. 2015 Apr 23;11(4):716–717. doi: 10.1080/15548627.2015.1017223

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© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC

© Charles Grose, Erin M Buckingham, Wallen Jackson, and John E Carpenter

This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 1. — Autophagosomes (green spheres) in the cytoplasm of cultured cells infected with VZV. VZV-infected monolayers were fixed with 2% paraformaldehyde, permeabilized with 0.02% Triton X-100, and then immunolabeled with a rabbit polyclonal antibody against LC3 and a mouse monoclonal antibody against the VZV major immediate-early protein IE62. Monolayers were observed with a Zeiss LSM710 Spectral confocal microscope using 63X high numerical-aperture oil immersion objective lenses. Confocal Z-stacks comprising 40 optical slices were reconstructed into 3D animations with the aid of Imaris software. The Spot function in Imaris automatically locates autophagosomes based on size and intensity thresholds; each autophagosome is represented by a sphere 500 nm in diameter. With this imaging technology, individual autophagosomes are easily detectable within infected cells. Note: VZV infection induces fusion of cells, thus there is a large cluster of nuclei in the image. Blue, Hoechst 33342 nuclear stain; red Alexa Fluor 546, VZV protein; green Alexa Fluor 488, LC3-labeled autophagosomes.