H 7100 tem

Transmission electron microscopy (TEM) was used for three purposes. First, TEM was used to verify the presence of intact, fully assembled virions from HSB2 and MOLT-3 host cells used for virus storage and propagation. Second, TEM was used to demonstrate the presence of HHV6 virus particles in differentiated human neural stem cells (dHNSC). Third, TEM was used to validate the presence of virus for qPCR-based titers from storage cells or dHNSC. For cell-free virus samples, the aforementioned preparation via sonication (or, alternatively, freeze-thaw cycles) was followed by concentration of virus suspensions (i.e., filtered lysates) using 30kDA MWCO spin concentrator (Sigma-Millipore). From the retentate, ~5μL of concentrated viral suspension was spotted onto a formvar-coated copper grid and incubated for 10 min in a humidity chamber. The sample was then rinsed and negatively stained with a 2% uranyl acetate solution for 30 sec before excess solution was wicked from the grid and allowed to air dry for 1 hr. Samples placed on grids, and after 2 to 10 minutes, 2% uranyl acetate was added to the grid. Grids were imaged with a Hitachi H-7100 TEM at 75 kV. Images were captured at 60,000– 200,000X magnification.

For TEM, inflorescences were harvested when plants were 4–8 cm tall. Tissues were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde in 0.05 M PB buffer for five hours at 4 °C, washed five times with 0.05 M PB buffer for 10 min at 4 °C, and fixed with OsO₄ buffer at 4 °C overnight. The resulting tissues were washed with 8% sucrose in water for two hours at 4 °C, dehydrated with an ethanol series, and infiltrated with Eponate 812 by incubating the samples at room temperature for several hours to overnight in increasing concentrations of resin. Then, the resin was polymerized in an oven at 60 °C for 48 h. Resin-embedded samples were sectioned to 70 nm widths with a diamond knife on a ultramicrotome. Sections were collected on a 0.5% formvar coated slot grid. Grids were post-stained for 5 min with 2% aqueous uranyl acetate and for 10 min with Reynolds lead citrate. Images were taken using a H-7100 TEM (Hitachi).

TEM was also used to image virus particles associated with host cells. These included both virus storage cells (HSB2 and MOLT-3 cells) and dHNSC infected with either virus. For virus-infected storage cells in or for virus-infected monolayers of dHNSC, cells were fixed with a 4% solution of paraformaldehyde (PFA). The samples were place on grids and then gently rinsed and negatively stained with a 2% solution of uranyl acetate. Samples were incubated for 2 hr at 4°C before rinsing with distilled water and wicking off excess solution from the grid and allowed to air dry for 1 hr. After air drying for 2 hr., samples were imaged with a Hitachi H-7100 TEM at 75 kV. Images were captured at 60,000– 200,000X magnification. Regions of the grid that showed virions blebbing from membrane or virus particles within the intracellular space were targeted for imaging. Detachment of HNSC from the surface substrate was required, to capture co-localization of virus particles within intact cells.