Leica uc7

Fresh sorghum embryo tissue of 1 mm³ in size was collected 36 h after germination, and transferred to an EP tube filled with a new electron microscope fixative for further fixing. Then, the processing method of the sample was referred to Nie et al. [32 (link)]. The samples were stained in 2% uranyl acetate saturated alcohol solution for 8 min in the dark. Sections were cut with an ultrathin slicer (LEICA UC7, Leica, Wetzlar, Germany). A transmission electron microscope (HT7700, Hitachi, Tokyo, Japan) was used for observation and image analysis.

Tissues were microwaved at full power (700W microwave oven) for 10 s to enhance penetration of the fixative through the depth of the slice with a final temperature of <35°C. Slices were left overnight in the same fixative and then rinsed with 0.1M phosphate buffer (4× for 15 min). First, they were immersed in 1% osmium for 1 hour and rinsed in buffer (3× for 15 min). Next, they were immersed in ascending concentrations of acetone (50, 70, 90, and 100%). Finally, they were quickly immersed in Spurr's resin at room temperature overnight and then embedded in coffin molds in Spurr's resin and cured for 8 hours at 70°C in an oven. They were then vibrasliced at 60 nm (Leica UC7, Leica). Ultrathin sections were counterstained with saturated aqueous uranyl acetate, followed by Reynolds lead citrate for 5 min. Sections were photographed with a transmission electron radiography (Hitachi H-7500, Hitachi) at 5,000× magnification.

Mitochondrial ultrastructure was assessed in several pieces obtained from different regions of the left ventricle that were pre-fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer, post-fixed in 2% osmium tetroxide (OsO4) in 0.1M sodium cacodylate buffer, dehydrated in an ethyl alcohol series, embedded with epoxy resin, and thermally polymerized as previously described (Katragadda et al., 2009 (link)). Ultra thin-sections (60 nm) were cut by an ultramicrotome (Leica UC7, Leica Microsystems Inc., ON, Canada) and then stained with 4% uranyl acetate and Reinold's lead citrate. The contrasted sections were imaged under a Hitachi H-7650 transmission electron microscope at 80 kV equipped with a 16 megapixel EMCCD camera (XR111, Advanced Microscopy Technique, MA, USA) was used for viewing the sections (Cho et al., 2007 (link); Katragadda et al., 2009 (link)).

C. albicans suspensions were adjusted to an initial concentration of 5 × 10⁴ cells/ml, and the suspensions were then transferred to new EP tubes, treated with different concentrations of EVs (0, 120 µg/ml), and cultured in KSFM (37°C, 5% CO₂) for 12 h. Each cell suspension was collected by centrifugation (12000 rpm, 10 min), and the pellet was then suspended and incubated at 4°C in electron microscope fixative for 4h. The samples were washed three times with calcium carbonate buffer solution and treated with 1% agarose solution. The agarose blocks with samples were post fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 2 h at room temperature while protected from light. After the OsO4 was removed, the tissues were rinsed in 0.1 M PB (pH 7.4) 3 times for 15 min each. Then, the samples were dehydrated with an alcohol and acetone gradient. The cells were then embedded and polymerized. Thin slices (60−80 nm thick) were obtained by using a diamond cutter (Daitome, Ultra 45°) on a Leica superslicer (Leica, Leica UC7). The samples were stained with a uranium acetate-saturated alcohol solution (8 min) and lead citrate (8 min).The cells were then analyzed and photographed using TEM (Hitachi, HT7800/HT7700). The integrated optical density (IOD) and thickness of C. albicans cell wall were quantified and analyzed using Image-Pro Plus image analysis software Version 7.0.1 (Media Cybernetics Inc).

On day 30, paraffin sections (5 mm × 5 mm) of the samples were taken, fixed with a formalin–acetic acid–alcohol fixative, dehydrated in an alcohol and xylene series, embedded in paraffin, cross-sectioned to a thickness of 10 μm, and red–solid green stained. The total thickness of the transverse sections, as well as the thickness of the upper epidermis, palisade mesophyll tissue, and spongy mesophyll tissue, was measured under a light microscope using a micrometer.
Pieces of the functional leaves were sampled (1 mm × 1 mm), quickly placed in a 2.5% glutaraldehyde fixative solution, and evacuated with a vacuum pump. After the pieces sank to the bottom of the fixative solution, they were maintained at room temperature (25 °C) for 2 h, and then transferred to a refrigerator and stored at 4 °C. The samples were rinsed three times with 0.1 M phosphate buffer (PB, pH = 7.4) for 15 min each, fixed with 1% citric acid in 0.1 M phosphate-buffered saline (pH = 7.4) at room temperature (25 °C) for 5 h, and rinsed again three times with 0.1 M PB (pH = 7.4) for 15 min each. The leaf tissue was sectioned on a dehydration-infiltration-embedding-slicer (Leica, LeicaUC7) and imaged using a section-staining-transmission electron microscope (HITACHI, HT7700).

Autophagy-related membrane structures were evaluated by transmission electron microscopy. The brains were cut into blocks (Leica UC7, Leica), fixed in stationary liquid (G1102, Servicebio), postfixed in 1% osmium tetroxide for 2 h, dehydrated in a graded series (20–100%) of ethanol, and finally polymerized with an 812 Embedding Kit (90529-77-4, SPI) for 48 h at 60 °C. Each block was observed under a transmission electron microscope (HT7700, Hitachi).