Jem 2200fs transmission electron microscope

The electron microscopic examination was performed following the procedure described by Peerapanyasut et al. [26 (link)] with slight modification. Briefly, pieces of renal cortical and liver tissues were fixed overnight with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4, 4 °C), postfixed in 2% phosphate-buffered osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon resin using EMbed-812 embedding kit (Electron Microscopic Sciences, PA, USA). Sections (60–80 nm) were mounted on copper grids, stained with uranyl acetate and lead citrate, and examined using a JEM-2200 FS transmission electron microscope (JEOL, Tokyo, Japan).

Transmission electron microscopy was used to examine the liver ultrastructure. A slight modification of previously published protocol [17 (link)] was applied. Liver tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 °C then subsequently post-fixed in 2% phosphate-buffered osmium tetroxide. After dehydration in graded ethanol, the tissues were washed in propylene oxide, and embedded in Epon resin using EMbed-812 embedding kit (Electron Microscopic Sciences, Hatfield, PA, USA). The obtained ultra-thin sections (60–80 nm thick) were mounted on copper grids, stained with uranyl acetate and lead citrate, and inspected with JEM-2200 FS transmission electron microscope (JEOL, Tokyo, Japan).

The exosome preparations were directly deposited onto glow-discharged C-flat 2/2-2C grids (Protochips, Morrisville, NC, USA). Grids were plunge-frozen in liquid ethane using a Vitrobot Cryo Fixation Device (FEI, Hillsboro, OR, USA). Grids of cryo-fixed preparations were imaged on a JEM-2200FS transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 200 kV and fitted with a DE-20 direct electron detector (Direct Electron LP., San Diego, USA). Zero-loss image stacks were recorded at −3 μm under focus, with an electron dose of ~5e/Å2 and a nominal magnification of ×25,000 and a pixel size of 0.23 nm. Stacks were aligned (using dose-weighting and patch options) using DE scripts (Direct Electron LP., San Diego, USA) and Motioncorr2 [13 (link)].

To study the cytopathology of H5N1-infected hNPCs, cells in 6-well microtiter plate were infected by a 1 MOI of H5N1 virus. At 24 hpi, cells were fixed in 2.5% glutaraldehyde for 48 hours, washed three times with phosphate buffer saline (PBS), and post fixed in 2% osmium tetroxide for 1 hour at RT. After being serially dehydrated in ethanol, the cells were incubated in propylene oxide and embedded in resin (EM-bed 812, Electron Microscope, Washington, USA). Semi-thin sections were cut and stained with toluidine blue for light microscope examination. Thereafter, ultra-thin sections were cut, mounted on copper grid, and electron contrasted with uranil acetate and lead citrate. The grids were examined and photos were taken under a JEM-2200FS transmission electron microscope (JEOL ltd., Tokyo, Japan).

High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) and high resolution transmission electron microscopy (HRTEM) images were obtained with a JEM-2200FS transmission electron microscope (JEOL Ltd., Japan, acceleration voltage 200 kV, lattice resolution – 1Å) equipped with a Cs-corrector and an EDX spectrometer (JEOL Ltd., Japan). The minimum spot diameter for the step-by-step line or mapping elemental EDX analysis was ∼1 nm with a step of about 1.5 nm. Some TEM micrographs were obtained with a JEM-2010 instrument (lattice resolution 1.4 Å, acceleration voltage 200 kV). Analysis of the local elemental composition was carried out by using an energy-dispersive EDX spectrometer equipped with Si(Li) detector (energy resolution 130 eV).