Em uc6 ultramicrotome

hDPSCs were cultured with 50 μg/ml MCD for 24, 48, or 72 h, fixed with 2% glutaraldehyde for 1 h at 4°C, then incubated in PBS overnight, and post-fixed in 1% osmium tetroxide in the same buffer for 2 h at room temperature. After dehydration with 50, 70, 90, and 100% ethanol, the samples were embedded in Epon. Ultrathin sections of cells were cut using a Leica EMUC6 ultramicrotome (Leica Biosystems, Wetzlar, Germany) and sections were settled on 200-mesh carbon-coated copper grids. After staining with 2% uranyl acetate and lead citrate, the samples were observed using an EM-14100BINC TEM (JEOL, Tokyo, Japan) at 80 kV. TEM images were taken to observe autophagic vesicles.

Sterile and dry filter paper (0.8 cm × 5.0 cm) was placed around the edge of a 4-day-old colony in PSA plates, and we added α-MG (500 μg·mL⁻¹), carbendazim (4.5 μg·mL⁻¹), or 0.5% DMSO solution (used as a control). After 24 h of treatment, the mycelium pellets (1 × 1 × 3 mm) below the paper were cut for the specimens. The specimens for TEM were processed as described by Liu et al. [43 (link)], with minor modifications. The samples were pre-fixed in 3% glutaraldehyde in 0.1 mol·L⁻¹ phosphate-buffered saline (PBS, pH 7.2) at 4 °C overnight. After rinsing three times with PBS for 10 min each time, the specimens were post-fixed with 1% w/v osmium tetraoxide solution for 2 h at room temperature and rinsed with PBS three times. The samples were dehydrated using a series of ethanol solutions in the order of concentration: 30, 50, 70, 80, 90, 95, and 100% for 15 min at each step. Afterwards, the specimens were embedded in Epon 812 and polymerize. Thin sections were cut by an EM UC6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and double-stained with uranyl acetate and lead citrate. The grids were observed using a HT7700 transmission electron microscope (Hitachi Company, Tokyo, Japan) operated at 60.0 kV.

To select cells in metaphase, resin-embedded samples were pre-inspected using an Axiolab RE upright brightfield microscope (Zeiss, Germany) with a 5 x and a 40 x objective lens (Zeiss, Germany). Selected cells in metaphase were sectioned using an EM UC6 ultramicrotome (Leica Microsystems, Austria). Ribbons of semi-thick (~300 nm) serial sections were collected on Formvar-coated copper slot grids, post-stained with 2% (w/v) uranyl acetate in 70% (v/v) methanol, followed by 0.4% (w/v) lead citrate (Science Services, USA) in double-distilled water. In addition, 20 nm-colloidal gold (British Biocell International, UK) was attached to the serial sections, serving as fiducial markers for subsequent electron tomography. The selected cells were then pre-inspected at low magnification (~2900 x) using either an EM906 (Zeiss, Germany) or a TECNAI T12 Biotwin (Thermo Fisher Scientific, USA) transmission electron microscope operated at either 80 or 120 kV, respectively.

For TEM, three specimens of B. townsendi (42, 46, 47 mm TL) were dissected immediately after collection while still on board the research vessel, and extracted ICs as well as pieces of intestinal wall were fixed in 2.5% glutaraldehyde in Sorenson’s phosphate buffer (pH 7.2). Fixed samples were held at 4°C for several days, then rinsed three times in buffer and postfixed in 1% OsO₄ in buffer for 1 h. After osmium treatment, they were rinsed three times in buffer, dehydrated through an ethanol series of ascending concentrations (to 100%), transferred to propylene oxide, infiltrated, and then embedded in Araldite 502 epoxy resin (Electron Microscopy Sciences, Hatfield, PA, United States). Ultrathin sections (70 nm) were obtained with a diamond knife using an EM UC6 ultramicrotome (Leica Microsystems). Sections were mounted on Formvar-covered copper grids, stained with uranyl acetate and lead citrate, and examined using an EM 10 CR transmission electron microscope (Carl Zeiss AG, Oberkochen, Germany). Electron micrographs were digitally recorded from phosphor imaging plates using a Micron imaging plate scanner (DITABIS, Pforzheim, Germany). Composite overview images were created using the software AutoStitch (Brown and Lowe, 2007 (link)).

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).