Embed 812 resin

HEK293T cells infected with SARS-CoV-2 at an MOI of 10 were prepared for electron microscopy. At 1 day post-infection, cells were fixed and embedded as previously described.^{98 (link)} Briefly, after washing with 0.1 M phosphate buffer, cells were post-fixed with 1% osmium tetroxide for 2 h at 4 °C. For dehydration, cells underwent an ethanol series (50%, 70%, 75%, 90%, 95% and 100%) and were subsequently placed in propylene oxide. Dehydrated cells were embedded in Embed-812 resins (Electron Microscopy Sciences, EMS) and polymerised at 70 °C for 24 h. They were sectioned at 80 nm using a Leica Ultramicrotome (Leica, Bensheim, Germany) with diamond knives and mounted on 150 mesh copper grids. The sectioned cells were post-stained with 2% uranyl acetate and 1% lead citrate. Finally, the cells were observed using conventional TEM (JEM-1400 Plus, JEOL, Tokyo, Japan) at 120 kV.

For TEM, zebrafish hearts were fixed in 2.5% fresh glutaraldehyde in 100 mM cacodylate buffer overnight at 4 °C, then washed in cacodylate buffer. Next, the hearts were post-fixed in 1% tannic acid, then 1% osmium tetroxide, dehydrated in acetone, and embedded in Embed-812 resin (Electron Microscopy Sciences, Hatsfield, PA, USA). Two-micron thick sagittal sections were cut and stained with toluidine blue for light microscopy analysis. Ultrathin sections (100 nm thick) were mounted on single-slot or 200-mesh copper or nickel grids and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, Oregon).

1- to 2-µm cubes (six cubes/animal) of tibialis anterior muscle were immersion fixed in 0.1 M sodium cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde and 20 mM calcium chloride. Tissues were post-fixed and heavy-metal-contrasted with potassium ferrocyanide, osmium tetroxide, thiocarbohydrazide, uranyl acetate and lead aspartate (Lafontant et al., 2013 (link)). After dehydration through an acetone series, tissues were embedded in Embed-812 resin (Electron Microscopy Sciences, Hatsfield, PA) and ultrathin sections (100 nm) were imaged on a Tecnai G2 Spirit BioTWIN (FEI Company, Hillsboro, OR) electron microscope. The abundance of triads was calculated by counting the number of triads per Z-line junction. At least 75 non-overlapping images were captured per animal at 26,500× from at least 15 individual fibers. Images were evenly spaced along the length of each fiber and no more than five images were captured per fiber.

Frozen PBMCs from participants were thawed and sorted memory CD45RO⁺CD3⁺CD4⁺ T cells (4 × 10⁶) were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour at room temperature. The cell pellets were then fixed for 2 hours at room temperature by immersion in freshly prepared triple-aldehyde DMSO (85 (link)–87 (link)). Cell pellets were postfixed in ferrocyanide-reduced osmium tetroxide. Another water rinse was followed by an overnight soak in acidified uranyl acetate. After again rinsing in distilled water, the sample blocks were dehydrated in increasing concentrations of ethanol, passed through propylene oxide, and embedded in EMBed 812 resin (Electron Microscopy Sciences). Thin sections were sequentially stained with acidified uranyl acetate followed by a triple lead stain (88 (link)). These sections were examined in a FEI Tecnai Spirit (T12) transmission electron microscope with a Gatan US4000 4k × 4k CCD. A total of 20 EMI sections from 2 independent experiments were analyzed to generate the data displayed in Figure 4.

Cells were fixed directly in culture medium with final concentrations of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) (overnight), washed three times in double-strength buffer followed by postfixation with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.2) (overnight), and washed again in the same buffer. The samples were further processed as described previously (71 (link)), except that samples were embedded in EMBed-812 resin (Electron Microscopy Sciences) and sections were examined using a JEM-1400Plus instrument (JEOL Ltd., Inc.). For peptidoglycan thickness measurements, 35 and 41 transmission electron microscopy (TEM) micrographs of the wild type and the kpsM mutant were used, respectively. Peptidoglycan thickness was measured in four points of the cell whenever possible.