Epon 812

Correlative FM-EM allows individual cells to be examined by both FM and EM. Tet-BZLF1/B95-8 cells were cultured on gridded 35-mm glass-bottom dishes (Mat Tek) coated with 1% gelatin in RPMI 1640 medium. To induce lytic EBV replication, doxycycline was added to the medium. After 24 h, cells on the grid were fixed with 4% paraformaldehyde solution in phosphate buffer (PB), stained with specific antibodies, and examined by CLSM. The same specimens were postfixed with 1% osmium tetroxide and 0.5% potassium ferrocyanide in PB for 1 h on ice, washed with distilled water (three times for 1 min each), dehydrated in an ethanol series (50, 70, 90 and 100% for 5 min each), and embedded in Epon-812 (TAAB Laboratories) at 65°C for 48 h. Ultrathin sections of cells were stained with saturated uranyl acetate and Reynold’s lead citrate solution. Electron micrographs were taken using a JEM-1400EX transmission electron microscope (JEOL). For EM, pcDNA-BZLF1 was transfected by electroporation into B95-8 cells to induce the lytic phase. The cells were harvested 24 h after transfection. After fixation, dehydration, and embedding, electron microscopic analysis was performed by using a JEM-1200EX transmission electron microscope at 80kV (JEOL).

Spinal cords, which were fixed by perfusion with 2% glutaraldehyde–2% paraformaldehyde buffered with 0.1 M PB (pH 7.2) and further immersed in the same fixatives for 2 h, were transversely sectioned into 1 mm blocks. Lumbar roots, treated in the same methods as spinal cords, were not sectioned before postfixation with 2% osmium tetroxide. Samples were postfixed with 2% osmium tetroxide buffered with 0.1 M PB (pH 7.2), dehydrated with a graded series of alcohol, and embedded in Epon 812 (Taab Laboratories Equipment, UK). One-micrometer sections were cut with an ultramicrotome Leica EM UC6 (Leica, Germany) and stained with toluidine blue.

Cellular samples were fixed in 0.9% NaCl, 1% glutaraldehyde (Wako) for 1 h at 4 °C. Cells were pelleted and re-suspended in 1 mL of a 37 °C pre-warmed 1.1% agarose solution (Bio-Rad). After centrifugation (3000 rpm at 37 °C), pellets were placed at 4 °C and cut into small specimens, washed in 5% sucrose PBS (Wako) and post-fixed with 5% sucrose, 1.5% osmic acid (Nisshin EM) in 100 mM phosphate buffer (Wako) pH 7.3, for 1 h at 4 °C. Post-fixed specimens were washed in 5% sucrose PBS and dehydrated in a graded series of ethanol (Wako) and propylene oxide (Wako), then embedded in epoxy resin EPON 812 (TAAB Laboratories). Ultrathin sections were obtained with an ultra-microtome (Leica EM UC7) and subjected to double staining with a uranyl-acetate (Wako) and lead-citrate (Nacalai Tesque). Samples were observed under a JEM-1400Plus Electronic Microscope (JEOL). TEM were analyzed by collecting images of at least 3 representative areas (RA) at magnification of ×500. One RA contained 10–20 nucleated cells. Then representative images were taken at higher magnifications.

Mouse heart specimens, which were harvested immediately after the mice were euthanized on day 7 after TAC surgery, were fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer for 2 h. The samples were washed with 0.1 m phosphate buffer, postfixed in 1% osmium tetroxide in the same phosphate buffer for 2 h, dehydrated in a graded series of ethanol washes, and embedded in Epon 812 (TAAB, Aldermaston, UK). Semi‐thin sections were sliced at 1 μm and stained with toluidine blue. Ultrathin 90‐nm sections were collected on copper grids, double‐stained with uranyl acetate and lead citrate, and then observed using transmission electron microscopy (H‐7011; Hitachi, Tokyo, Japan).

The ultrastructure of cells and EVs was observed by transmission electron microscopy as previously described^{8 (link)} with some modifications. Briefly, sorted cells and EVs were fixed with 2.5% glutaraldehyde (Nacalai Tesque), 2% paraformaldehyde (Wako) in 0.1 M phosphate buffer (pH 7.4) at 4 °C overnight. Cells were then dehydrated and embedded in Epon 812 (TAAB Laboratories). Ultrathin sections were obtained from the Epon blocks and stained with uranyl acetate and lead citrate. For negative staining of EVs, a grid with a supporting film was placed on a drop of the EV suspension for 15 min and excess solution was removed with a filter paper. The grid was then placed in 2% uranium acetate aqueous solution for 2 min, followed by air-drying. Sections and stained EVs were observed under an electron microscope (H-7650, Hitachi).

Cells incubated in darkness, normal or high light for 6 hours, as well as gametic cells and zygotes from the crosses were concentrated, deposited on polylysine-coated cover slips, fixed with a mixture of 2% (wt/vol) paraformaldehyde, 1% (wt/vol) glutaraldehyde in 0.2 M phosphate buffer (PB), pH 7.4, post-fixed with 1% (wt/vol) OsO4 supplemented with 1.5% (wt/vol) potassium ferrocyanide, dehydrated in ethanol, and embedded in epon 812 (TAAB Laboratories Equipment). Ultrathin sections were prepared with a Reichert UltracutS ultramicrotome (Leica), counter-stained with uranyl acetate, and viewed at 80kV with a Transmission Electron Microscope (TEM; Tecnai Spirit G2; Thermo Fisher Scientific, Eindhoven, The Netherlands) equipped with a QUEMESA CCD 4K camera (EMSIS GmbH, Münster, Germany) using iTEM software (EMSIS).

The differentiated IEC#17 cells were prepared in 2.5% Matrigel-coated Transwell membranes (Corning, NY, USA: 3470) or Cell Desk LF (Sumitomo Bakelite, Tokyo, Japan). The cells were infected with SARS-CoV-2 at an MOI of 0.1. At 24 hpi, the cells were fixed with 2% formaldehyde and 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.4) and washed three times with the same buffer for 5 min. Cells were post-fixed for 1 h with 1% osmium tetroxide and 1% potassium ferrocyanide in a 0.1 M sodium phosphate buffer (pH 7.4), dehydrated using a graded series of ethanol, and embedded in Epon812 (TAAB, Berks, UK). Ultra-thin Sects. (80 nm) were stained with saturated uranyl acetate and a lead citrate solution. Electron micrographs were obtained with a JEM-1400plus transmission electron microscope (JEOL, Tokyo, Japan).