Jem 1400ex

Cells cultured on coverslips were fixed with 2% formaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and post-fixed in a mixture of 1% osmium tetroxide and 1.5% potassium ferrocyanide in the same buffer. After ethanol dehydration, samples were embedded in epoxy resin. Ultrathin sections were observed with an electron microscope (JEM1400EX, JEOL) operated at 80 kV.

At 7 days after surgery, nerve-injured segments were harvested and fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment Ltd., Reading, Berkshire, United Kingdom) overnight at 4 °C. The nerve segments were subsequently fixed in 2% osmium tetroxide (OsO₄; TAAB Laboratories Equipment Ltd.) for 2 h, separately dehydrated in an ethanol gradient (50%, 70%, 80%, 90%, 95% and 100%), and treated in a gradient of EPON812 (33%, 50%, 66% and 100%; TAAB Laboratories Equipment Ltd.) in propylene oxide (Nacalai Tesque, Inc., Kyoto, Japan). Tissues were embedded in EPON812 in a 60 °C oven for 48 h. Semi-thin sections (200 μm) were cut vertically with an ultramicrotome (Ultracut S; Leica Microsystems, Wetzlar, Germany), stained with 1% toluidine blue solution, and examined under a light microscope (BZ9000; Keyence). The density of the myelinated fibers (fibers/1000 mm²) was analyzed in five non-overlapping visual fields per specimen. Ultrathin sections (70–80 nm) were cut with an ultramicrotome. We chose axons exhibiting an equivalent diameter and evaluated the G-ratio as the ratio of the inner axonal diameter to the total outer diameter. The stained samples were observed under TEM (JEM-1400EX; JEOL Ltd., Tokyo. Japan). We randomly selected five separate fields per slice for analysis.

Cells were cultured on coverslips coated with 150 μm grids (Matsunami Glass Ind.). The cells were stimulated with DMXAA (25 µg ml⁻¹) in the presence of protease inhibitors (E64d (30 µg ml⁻¹) and pepstatin A (40 µg ml⁻¹)) and orlistat (20 µg ml⁻¹). Cells were fixed with 2% PFA–2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min at room temperature and rinsed three times for 15 min each time in 0.1 M phosphate buffer (pH 7.4). The fluorescence images were obtained using a confocal microscope (LSM880 with Airyscan (Zeiss)). They were fixed again with 2% PFA–2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for more than 15 min at 4 °C, and then with a reduced osmium fixative. After embedding in Epon812 resin, areas containing cells of interest were trimmed according to the light-microscopic observations, and serial ultrathin sections (80 nm thickness) were prepared and observed with an electron microscope (JEM1400EX; JEOL)^{50 (link),51 (link)}.

Eyes were collected from 2-year-old normal and CSNB dogs after euthanasia. Following removal of anterior segment, the retinas were fixed in 2.5% glutaraldehyde for 2 hrs. They were subsequently fixed in 1% osmium tetroxide for 90 min, and then dehydrated through a graded series of ethanol (50–100%), and cleared in propylene oxide. Finally, the tissues were embedded in epoxy resin. Ultrathin sections were cut on an ultramicrotome (Ultracut E; Reichert-Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate. The stained sections were observed by transmission electron microscopy (JEM-1400EX; JEOL Ltd., Tokyo, Japan).

Kidney tissues were fixed in formalin, embedded in paraffin, cut into 4 μm sections, and stained with Periodic acid-Schiff reagent. For immunofluorescence, the sections of kidney tissues were embedded in OCT compound, frozen, cut into 4 μm sections, and stained with anti-IgG (1:100) (115-095-166, Jackson Immuno Research, West Grove, PA), anti-IgM (1:100) (1020-02, SouthernBiotech, Birmingham, AL), and anti-C3 (1:3200) (55500, MP Biomedicals, Irvine, CA) antibodies. For transmission electron microscopy, kidney tissues were double-fixed in 2.5% glutaraldehyde and 2% osmium tetroxide. The tissues were embedded in Epon 812. Ultrathin sections were generated using an ULTRACUT S (Leica, Germany) and double-stained with uranyl acetate and lead. Images were obtained using a transmission electron microscope (JEM-1400 Ex; JEOL, Tokyo, Japan) at 60 kV.

Freshly isolated EVs suspensions were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). A drop of each sample was placed on a carbon-coated copper grid for 20 min and negative staining with 1% uranyl acetate for 1 min. The morphology of EVs was observed under the transmission electron microscope (JEM-1400EX, JEOL Ltd., Tokyo, Japan).

For conventional EM, primary hepatocytes were fixed in 0.1 M PB containing 2% paraformaldehyde and 2% glutaraldehyde, followed by a process of the reduced-osmium method^{66 (link)}, embedding in Epon812, sectioning, and staining with uranyl acetate and lead citrate. For immune-EM, the cells were fixed in 0.1 M PB containing 4% paraformaldehyde and 4% sucrose, and then frozen after infusion with 2.3 M sucrose and 20% polyvinylpyrrolidone^{67 (link)}. Ultrathin sections were mounted on Formvar carbon-coated nickel grids, blocked with 1% bovine serum albumin (BSA) in PBS, incubated with anti-p62 (GP62-C, Progen) antibody, and then incubated with colloidal gold (6 nm)–conjugated goat anti-guinea pig antibody (Jackson ImmunoResearch laboratories, Inc). The sections were observed with an electron microscope (EM; JEM1400EX; JEOL)