Leaf properties were observed and investigated using the methodologies of Yan and Wang (2017) and Hao et al. (2019) [1 ,30 (link)]. Each cultivar’s uppermost first leaf was collected at the four-leaf stage. The obtained samples were prefixed with 2% glutaraldehyde overnight. The prefixed samples were washed in phosphate buffer (PBS, pH = 7.4) the next morning, then postfixed in 2% OsO4 for 1 h before being washed in PBS at pH 7.4. The samples were dehydrated in a series of gradient alcohol concentrations before embedding in Epon 812 resin (SPI Supplies, Structure Probe, Inc., West Chester, Pennsylvania, The United States of America). Following a semi-thin section (3–4 µm), the leaf structures were photographed using an inverted phase contrast microscope equipped with a Leica DFC 295 imaging system (Leica DM IRB, Leica Microsystems, Wetzlar, Germany). The thickness of the upper epidermis, as well as the quantity and length of trichomes, were measured using the Leica application suite (LAS, Leica Microsystems, Wetzlar, Germany). Ten leaves from ten plants were inspected per replicate for each cultivar, with three replicates carried out.
Epon 812 resin
Manufactured by SPI Supplies
Sourced in United States
Epon 812 resin is a low-viscosity epoxy resin commonly used in laboratory applications. It is a primary component for embedding and sectioning specimens for microscopy and other analytical techniques. The resin cures to a hard, durable material that provides structural support and preservation of sample integrity.
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5 protocols using epon 812 resin
1
Leaf Microstructural Analysis of Cultivars
2
Kidney Tissue Ultrastructural Analysis
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Biopsy from kidney tissues were cut into pieces (1mm×1 mm×5 mm ), and fixed with glutaraldehyde at 4°C for 2 h. After wash with PBS, they were incubated with 1% osmium tetroxide at room temperature for 1 h, followed by dehydration with 30%--100% ethanol gradient. Next, the samples were infiltrated in 1:1 mixture of acetone and Epon 812 resin (SPI Supplies, West Chester, USA) overnight. Tissues were then embedded in araldite, sectioned with a diamond knife, stained with 1% uranyl acetate and 1% lead citrate, and examined with a transmission electron microscope (JME-1220; JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.
3
Transmission Electron Microscopy of HepG2 Cells
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The HepG2 cells were fixed in PBS with 2.5% glutaraldehyde, pH 7.4, post-fixed in 1% osmium tetroxide, followed by en bloc staining with 2% uranyl acetate. Samples were dehydrated in succession by adding ethanol concentrations and embedded in Epon 812 resin (SPI Supplies, West Chester, PA, USA). Sections were performed using a Tecnai 12 Biotwin TEM (FEI, Eindhoven, The Netherlands).
4
Ultrastructural Analysis of Autophagosome-Enriched DRibbles
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As mentioned earlier, SCC7 cells were treated for 24 hours to collect DRibbles. The autophagosome-enriched DRibble suspension was collected in 1.5 mL Eppendorf tubes and centrifuged at 12,000 g for 15 minutes. The supernatant was removed carefully, and 2.5% cold glutaraldehyde was added to fix samples at 4°C overnight. The next day, the samples were fixed in 1% osmium tetroxide for 1 hour after being rinsed, dehydrated in gradient acetone, replaced with 1:1 acetone:Epon 812 resin overnight, infiltrated with 100% Epon 812 resin (SPI Supplies, West Chester, PA, USA) for 1 hour, and embedded in Epon 812 resin. After polymerization for 24 hours at 60°C, ultrathin sections (70 nm) were cut with an LKB-V ultramicrotome (LKB, Bromma, Sweden) and stained with uranyl acetate and lead citrate. Then, the sections were observed with a JEM-1011 transmission electron microscope (JEOL, Tokyo, Japan), and images were acquired with a charge-coupled-device CCD camera (SIS, Münster, Germany).
5
Locating Intracellular Calcium using Potassium Pyroantimonate
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Potassium pyroantimonate is often used to detect and locate intracellular Ca2+ because it reacts with it to form deposits with high electron densities (Tian et al. 1998 ).
Small blocks of tissue (0.5×0.5×1 mm) containing secretory cavities at different stages of development were fixed in 0.1 M KH2PO4 buffer solution (pH 7.8) with 2.5% paraformaldehyde (v/v) containing freshly added 2% potassium pyroantimonate. The samples were washed five times with buffer (fresh buffered 2% potassium pyroantimonate) for 20 min each, post-fixed for 10 h at 4 °C in 2% (w/v)-buffered OsO4 containing 1% potassium pyroantimonate, washed five times in buffer without potassium pyroantimonate for 20 min each, dehydrated in a graded ethanol series (15%, 30%, 45%, 60%, 70%, 80%, 90%, and 100%), infiltrated, and then embedded in Epon 812 resin (SPI Supplies). A Leica EM UC7 microtome was used for cutting sections to a thickness of 70–80 nm. After staining with uranyl acetate and lead citrate, the sections were examined and photographed using a Philips Fei-Tecnai 12 TEM (Zheng et al., 2014 ). As additional controls, sections were incubated in 200 mM EGTA (pH 7.9) for 1 h (Tian et al., 1998 ).
Small blocks of tissue (0.5×0.5×1 mm) containing secretory cavities at different stages of development were fixed in 0.1 M KH2PO4 buffer solution (pH 7.8) with 2.5% paraformaldehyde (v/v) containing freshly added 2% potassium pyroantimonate. The samples were washed five times with buffer (fresh buffered 2% potassium pyroantimonate) for 20 min each, post-fixed for 10 h at 4 °C in 2% (w/v)-buffered OsO4 containing 1% potassium pyroantimonate, washed five times in buffer without potassium pyroantimonate for 20 min each, dehydrated in a graded ethanol series (15%, 30%, 45%, 60%, 70%, 80%, 90%, and 100%), infiltrated, and then embedded in Epon 812 resin (SPI Supplies). A Leica EM UC7 microtome was used for cutting sections to a thickness of 70–80 nm. After staining with uranyl acetate and lead citrate, the sections were examined and photographed using a Philips Fei-Tecnai 12 TEM (Zheng et al., 2014 ). As additional controls, sections were incubated in 200 mM EGTA (pH 7.9) for 1 h (Tian et al., 1998 ).
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