H 7500 transmission electron microscope

Fluorescence detections were carried out on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, United States, https://www.agilent.com). Transmission Electron Microscope (TEM) image, Nanoparticle Tracking Analysis (NTA), the SDS-PAGE and gel imaging analysis for the characterization of tumor cell-derived exosomes was supported by H-7500 transmission electron microscope (Hitachi High-Technologies Co., Japan, https://www.hitachihightech.com), ZetaView (Particle Metrix, Germany, https://www.particle-metrix.com), electrophoresis analyzer and ChemDoc XRS (Bio-Rad, United States, https://www.bio-equip.com), respectively.

For phenotype characterization, plants grown on MS medium were photographed by using an anatomic microscope. The small leaf segments were collected from 7- and 14-day-old Col-0 and mterf6-5 mutant plants and from 14-day-old complemented plants grown on MS medium (with/without 2% sucrose) under normal culture conditions. Later, a Hitachi H7500 transmission electron microscope (Hitachi High-Technologies, Tokyo) was used to observe the ultrastructure of chloroplasts^{73 (link)}. For generation of transgenic plants, the 2004-bp MTERF6.1 genomic sequence before the stop codon of MTERF6.1 was amplified with the primer set MTERF6-Sac-F/MTERF6.1-Sal-R and cloned into the pCAMBIA1300 vector, following FLAG and 39-Nos sequences. Furthermore, the genomic sequences of MTERF6.2 (2375 bp) and MTERF6.3 (2495 bp) were cloned for construction by using the same method (for primer design, see Supplementary Table S1). The constructs were transformed into MTERF6/mterf6-5 heterozygous plants by Agrobacterium-mediated transformation^{39 (link)}. These transgenic plants (pMTERF6::MTERF6-FLAG in the mterf6-5 mutant background) were screened and identified by using the same method as for the complementation lines mentioned above.

As there was a limitation to the electron microscopic analysis of SARS-CoV-2 from the perspective of pathogen safety management, bovine coronavirus (BCoV), which belongs to the genus Betacoronavirus like SARS-CoV-2, was used as a surrogate virus for the experiment. BCoV and SARS-CoV-2 share similar structures, and their S protein epitopes show a degree of homology [27 (link)]. Purified BCoV was incubated with nine volumes of either neutral ASE2 or UPW for 6 h; then, the samples were prepared for electron microscopy on 400-mesh carbon-coated collodion grids (NISSHIN EM Co., Ltd., Tokyo, Japan), in accordance with a two-step protocol [28 (link)]. The virus was negatively stained for 2 min with 2% phosphotungstic acid (pH 6.5), and the grids were examined using an H7500 transmission electron microscope (Hitachi High-Technologies Co., Tokyo, Japan). To evaluate the number of intact virions in the UPW- and neutral ASE2-treated groups, 0.52 µm² fields (20 in number) were randomly chosen for three samples of each group, and the total number of intact virions was counted per 20 fields for each sample. Virions with discernible S proteins, a well-defined, undisrupted envelope, and a uniformly clear color showing no staining agent penetration were judged as intact virions.

Formalin-fixed brain tissue from the brainstem of Case 1 was cut into 1-mm cubes, fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), and then post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2) at 4°C for 2 hours. Tissues were then dehydrated through a graded series of ethanol, replaced with QY-1 solution (Nisshin EM, Tokyo, Japan), and embedded in an epoxy resin (Quetol 651, Nisshin EM, Tokyo, Japan). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-7500 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan).

Samples of liver were fixed in 4% Formaldehyde/1% Glutaraldehyde, washed in Millonig’s Phosphate buffer and transferred to a Leica EMTP automatic tissue processor (Leica Microsystems). Tissues were post-fixed in 2% Osmium Tetroxide and processed into Agar 100 Epoxy resin (Agar Scientific). Samples were embedded and allowed to polymerise overnight at 60 °C. From the resulting blocks semi-thin (1 μM thick) sections were cut on a Leica UC6 Ultra-microtome (Leica Microsystems) and stained with Toluidine Blue to locate the correct region using light microscopy. Subsequently Ultra-thin (80-90 nm thick) sections were cut and contrasted using 2% Lead Citrate and Uranyless stain (TABB). Grids were examined using a Hitachi H7500 Transmission Electron Microscope (Hitachi High-tech) operating at 80 kV and images taken on a Gatan OneView Digital Camera (Gatan, Inc.). Mitochondrial number was quantified in images taken at × 7000 magnification through a manual count, corrected to image area.

Visualization of pseudovirus particles was performed on sections of virus-producing HEK293 cells. Briefly, cells were grown on sterilized 18 mm diameter coverslips cells and were transfected with 0.83 μg of SG3^Δenv pDNA (as described above) once cells had reached 70% confluency. Eight hours after transfection, the media were replaced, and 24 h after transfection the media were removed from the coverslips and the cells were washed with PBS. Samples were subsequently fixed with 2.5% glutaraldehyde in 0.1 M PB for 2 h at room temperature followed by an overnight 4 °C incubation before TEM processing. For EM processing, samples were washed 3 times with 0.1 M cacodylate buffer (pH 7.3) before being reduced with 1% OsO4 in cacodylate buffer and stained with 4% uranyl acetate. After staining, samples underwent ethanol dehydration and infiltration in Quetol–Spurr resin before polymerization in fresh resin at 70 °C for 2 days. Samples were then sectioned at 80 nm before staining on copper grids with uranyl acetate and lead citrate. After processing, samples were imaged on a Hitachi H-7500 transmission electron microscope (TEM, Hitachi High-Technologies, Fukuoka, Japan) equipped with a Megaview G2 CCD camera (Olympus, Toronto, ON, CA). Images were acquired from areas of free virions (i.e., virions that were not cell-associated) to validate our pseudovirus particle production methods.