Journal: Journal of Virology
Article Title: Polarized Cell Migration during Cell-to-Cell Transmission of Herpes Simplex Virus in Human Skin Keratinocytes
Figure Lengend Snippet: Ultrastructural characterization of HSV plaques in HaCaT cells. Monolayers of HaCaT cells in 35-mm dishes were infected with 100 PFU of HSV-1/well and processed at 24 h postinfection for TEM as described in Materials and Methods. (a) Low-power binocular image of the vertical face of the embedded resin block. This image gives a three-dimensional view along the horizontal surface of the plastic support. Arrows indicate the plaques of clustered piled-up cells, with the size of the arrow indicating the distance from the block front. (b) The block was then sectioned into semithin survey sections (500 to 1,000 nm) and stained with crystal violet. Images were collected on a Zeiss Axiovert 200M using a ×40 objective lens, and the panels were assembled using Adobe Photoshop. (c) Sequential ultrathin sections (50 to 70 nm) of the same sample were then obtained and collected onto slot grids, stained, and examined by TEM. Because the sections analyzed are immediately adjacent to the section stained with crystal violet, corresponding areas could be matched. The TEM images of the sections in panel c, sections 1 to 4, therefore correspond to the boxes in panel b, sections 1 to 4. Upper panels (lower magnification) and lower panels (higher magnification on inset area) show different features of infection within the plaque as discussed in the text. In the lower panels, the features include extracellular virions (black arrows with white outline), nucleocapsids (short black arrows), and cell tight junctions (white arrows). In the upper part of panels 3 and 4, cells with no distinct features of infection (e.g., no early chromatin marginalization) are indicated by long black arrows. Box 5 in panel b shows flat uninfected cells that correspond to the elongated cells (TEM data not shown).
Article Snippet: The sample was sectioned again when appropriate transverse sections were observed, and ultrathin sections (typically 50 to 70 nm) were cut and collected onto slot grids, stained with Reynold's lead citrate examined in a FEI Tecnai electron microscope with CCD camera image acquisition.
Techniques: Infection, Transmission Electron Microscopy, Blocking Assay, Staining