Jem 1011 transmission electron microscope

Parasites (2 × 10⁷) grown under different glucose conditions were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 at RT, treated with 1% (w/v) osmium tetroxide, and dehydrated with increasing concentrations of ethanol. Samples were transferred to propylene oxide and then successively to propylene oxide/LR-White resin mixtures at ratios of 1:1 and 2:1, embedded in LR-White resin, and polymerized at 56 °C overnight. Thin sections (60 nm) were obtained and incubated overnight at RT with the Mα-TvLEGU-2pep (mouse, 1:10) primary antibody, washed, and incubated with gold-conjugated anti-mouse IgG (1:60 dilution using 15 nm gold particles) as a secondary antibody (Ted Pella Inc., Redding, CA, USA). For double labeling, the sections were incubated with Mα-TvLEGU-2pep/RαTvPFO50r and Mα-TvLEGU-2pep/RαrTvAtg8b primary antibody combinations (both at 1:10 dilution). gold-conjugated anti-mouse IgG (1:60 dilution; 15 nm gold particles) and anti-rabbit IgG (1:60 dilution; 30 nm gold particles) were used as secondary antibodies (Ted Pella Inc.). The samples were counterstained with uranyl acetate and lead citrate. Samples incubated with PI serum or only the secondary antibodies were used as negative controls. A JEOL JEM-1011 transmission electron microscope was used (JEOL Ltd., Tokyo, Japan).

Fecal samples from the 2021 diarrhea outbreak (n = 16) were diluted to 10% in deionized water and separated into two aliquots. The first aliquot was clarified by a 2 min centrifugation at 12,000× g and a formvar, carbon-coated, 400-mesh copper grid was floated on a 50 µL drop of the supernatant for 15 min. Excess supernatant was removed from the grid with filter paper and stained with 3% aqueous phosphotungstic acid, pH 7.0, for 1 min. The second aliquot was first centrifuged at 1000× g for 15 min and the supernatant was subsequently centrifuged at 10,000× g for 30 min and 40,000× g for 1 h. The supernatant was discarded, and the pellet was resuspended in purified deionized water and a 50 µL drop of the resuspension was stained as indicated above. Grids were viewed with a JEOL JEM-1011 Transmission Electron Microscope at an accelerating voltage of 100 kV (JEOL USA, Inc., Peabody, MA, USA). Representative viral particles were digitally imaged using an XR80M Wide-Angle Multi-Discipline Mid-Mount CCD Camera from AMT (Advanced Microscopy Techniques, Woburn, MA, USA).

Oral proliferative tissue and lip skin samples were immediately fixed in 4% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2–3 h. They were washed (20 min 5 times) and post fixed in 1% OsO4 in the same buffer for 1 h. They were washed again in 0.1 M phosphate buffer (pH 7.4) and then dehydrated in graded alcohol and embedded in Agar Low Viscosity Resin (Agar Scientific Limited, Essex, England). Semi-thin section (400 nm) were cut on an EM UC6 ultramicrotome (Leica Microsystems) and were stained with 1% toluidine blue in water solution and examined by Leica DM 4000 B optical microscope. Ultra thin sections (60–70 nm), obtained from chosen areas, were collected onto 300-mesh grids coated with formvar and counterstained with lead citrate and uranyl acetate. The sections were observed with a JEOL JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a thermionic tungsten filament and operated at an acceleration voltage of 100 kV. Images were taken using a Morada cooled slow-scan CCD camera (3783 × 2672 pixels) and micrographs were taken with iTEM software (Olympus Soft Imaging System GmbH, Munster, Germany).