The exosomes obtained by centrifugation of 400 mL of the medium at high-speed were fixed in 2% glutaraldehyde overnight at 4 °C. Then, exosomes were fixed with 1% OsO4 for 1 h, dehydrated in ethanol, and finally embedded in resin. The embedded sample was sliced using a microtome and saturated sodium periodate and 0.1 N hydrochloric acid were each added onto the sections. After 10 min, the sections were observed under a TEM (H-500, HITACHI, Tokyo, Japan).
H 500
Manufactured by Hitachi
Sourced in Japan
The H-500 is a high-performance laboratory equipment designed for a variety of applications. It is a highly versatile and precise instrument capable of performing various analytical and experimental tasks. The core function of the H-500 is to provide accurate and reliable data for researchers and scientists in various fields.
Automatically generated - may contain errors
Lab products found in correlation
Colorview 2 camera, by Olympus (3 mentions)
Bx51 microscope, by Olympus (3 mentions)
Araldite, by Merck Group (2 mentions)
Epon 812, by Merck Group (2 mentions)
Bx60 light microscope, by Olympus (2 mentions)
Ultracut uct25 ultramicrotome, by Leica camera (2 mentions)
Jem 1400, by JEOL (2 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Methylene blue, by Merck Group (1 mentions)
Epoxy embedding medium kit, by Merck Group (1 mentions)
Em uc6 ultramicrotome, by Leica camera (1 mentions)
Uranyl acetate, by Polysciences (1 mentions)
Lead citrate, by Merck Group (1 mentions)
Formvar 15 95e, by Merck Group (1 mentions)
H 7000 electron microscope, by Hitachi (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Te300, by Nikon (1 mentions)
Tunel apoptosis assay kit, by Keygen Biotech (1 mentions)
Ultracut em uc7 ultramicrotome, by Leica camera (1 mentions)
Uhr fe sem su 8010, by Hitachi (1 mentions)
22 protocols using h 500
1
Exosome Isolation and Electron Microscopy
2
Ultrastructural Analysis of Ovary Development
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Ovaries and ovules at different stages of development were fixed for 24 h in 2.5% glutaraldehyde (Merck, Darmstadt, Germany) prepared on 0.1 M Sorensen’s phosphate buffer (pH 7.2) and containing 1.5% sucrose. Then samples were washed, post-fixed in 1% OsO4 (Sigma-Aldrich, St. Louis, Missouri, USA), dehydrated in ethanol of increasing concentrations (30, 50, 70, 96, and 100%), propylene oxide and embedded in a mixture of Epon-812 and Araldite (Merck, Darmstadt, Germany), according to the standard protocol.
For light microscopy, semi-thin sections (1–2 μm) were prepared using glass knives and an ultramicrotome LKB-V (LKB, Sweden), placed on glass slides, stained with 0.1% methylene blue (Merck, Darmstadt, Germany), and embedded in epoxide resin. Samples were analyzed and photographed using an Olympus BX51 microscope (Olympus, Shinjuku, Tokyo, Japan) supplied with a Color View II camera (Soft Imaging System, Münster, Germany).
For electron microscopy, samples were sectioned with a diamond knife using an ultramicrotome LKB-V (LKB, Sweden), placed on formvar-coated grids, and stained with uranyl acetate and lead citrate. Then thin sections were analyzed and photographed using an electron microscope H-500 (Hitachi, Ibaraki, Japan) and JEM-1400 (Jeol, Akishima, Tokyo, Japan).
For light microscopy, semi-thin sections (1–2 μm) were prepared using glass knives and an ultramicrotome LKB-V (LKB, Sweden), placed on glass slides, stained with 0.1% methylene blue (Merck, Darmstadt, Germany), and embedded in epoxide resin. Samples were analyzed and photographed using an Olympus BX51 microscope (Olympus, Shinjuku, Tokyo, Japan) supplied with a Color View II camera (Soft Imaging System, Münster, Germany).
For electron microscopy, samples were sectioned with a diamond knife using an ultramicrotome LKB-V (LKB, Sweden), placed on formvar-coated grids, and stained with uranyl acetate and lead citrate. Then thin sections were analyzed and photographed using an electron microscope H-500 (Hitachi, Ibaraki, Japan) and JEM-1400 (Jeol, Akishima, Tokyo, Japan).
3
Ultrastructural Analysis of N. davidi
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Adult specimens of N. davidi were decapitated and fixed with 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.4, 4°C, 2h), postfixed in 2% osmium tetroxide in a 0.1 M phosphate buffer (4°C, 1.5 h) and dehydrated in a graded series of concentrations of ethanol (50, 70, 90, 95 and 4x100% each for 15 min) and acetone (15 min). Afterwards, the material was embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma). Semi- (0.8 μm thick) and ultra-thin (70 nm) sections were cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thin sections were stained with 1% methylene blue in 0.5% borax and observed using an Olympus BX60 light microscope. After staining with uranyl acetate and lead citrate, ultra-thin sections were examined using a Hitachi H500 transmission electron microscope.
4
Ultrastructural Effects of Insecticidal Toxins on Midgut Cells
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Third instar of larvae H. armigera strain LF were starved for 24 h, then fed with 1.2 µg/g of Vip3AcAa, 1.2 µg/g of Cry1Ac toxin, or 0.01 M phosphate-buffered saline (PBS; as control) in an artificial diet for 3, 6, 12, 24, 36, and 48 h. The larvae were kept on ice for 15 min to immobilize them. Subsequently, the larvae were killed, and the midguts were excised and fixed in 2.5% w/v glutaraldehyde in 0.1 M phosphate buffer (PB) for 1 h at room temperature. Samples were washed three times with PB and post-fixed with 1% v/v osmic tetroxide in PB. Samples were dehydrated in a graded ethanol series, then rinsed in 100% acetone and embedded in epoxy resin 618 (Hitachi). Embedded tissues were sectioned using a Leica EM UC6 ultramicrotome, and double-stained with uranyl acetate and lead citrate. Images were captured via transmission electron microscopy (TEM) at an accelerating voltage of 80 kV (Hitachi, H-500) (Zhang et al. 2012b ).
5
Transmission Electron Microscopy of Desiccated and Rehydrated Specimens
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Ten desiccated (6 from the experimental and 4 from the control group) and ten rehydrated (6 from the experimental and 4 from the control group) specimens were prepared for analysis with a transmission electron microscope (Hitachi H500 at 75 kV) as described earlier (see I. Non-experimental analyses of storage cells in desiccated and hydrated specimens, light and transmission electron microscopy).
6
Transmission Electron Microscopy of VLPs
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Samples were prepared as described previously (22 (link)). Briefly, purified VLPs were fixed in Karnovsky solution and loaded onto copper grids coated with a support film (Formvar 15/95E; Sigma-Aldrich, St. Louis, MO, USA). After drying, the material was stained with uranyl acetate (Polyscience, Inc., Warrington, PA, USA) and lead citrate (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the grids were washed with water and dried in air at room temperature. Ultrastructural observations were performed by using a Hitachi H500 transmission electron microscope at an accelerating voltage of 75 kV.
7
Purification and Characterization of A/PR-8 Virus
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A/PR-8 virus was purified by centrifugation at 142,190 ×g for 4 h along a 10–50% (w/w) sucrose density gradient and treated with concentrated Melia extract for 120 min at 37 °C. Samples were then stained with 2% phosphotungstic acid and examined by electron microscopy (H500; Hitachi, Japan). For comparison, untreated virus was purified along a 10–50% (w/w) sucrose density gradient by centrifugation at 142,190×g for 4 h in an SW 50.1 rotor, and buoyant density was determined by centrifugation at 142,190×g for 4 h along the 10–50% (w/w) sucrose density gradient. Morphological observation of the virus particles was performed using a Hitachi H7000 electron microscope, as described previously [9 (link)].
8
Grain Sample Preparation for Microscopic Analysis
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Selected samples of grain were fixed using a 2.5% glutaraldehyde solution in 0.1 M Na2HPO4/K2HPO4 Sorensen buffer, pH 7.3, supplemented with sucrose (15 mg/mL), for one day at 4 °C. After washing from the fixing mixture at the same buffer, the samples were post-fixed with a 1.0% solution of osmium tetroxide (OsO4) for 1 h, then dehydrated in increasing concentrations of ethanol (30, 50, 70, 96 and 100%), propylene oxide and encapsulated with Epon–Araldit epoxy resin kit. For light microscopy, semi-thin sections (1–2 μm) were prepared using glass knives and an ultramicrotome LKB-III (LKB, Bromma, Sweden), placed on glass slides, then stained with 0.1% toluidine blue (Merck, Darmstadt, Germany) and embedded in epoxide resin. Samples were analyzed and photographed using an Olympus BX51 microscope (Olympus, Shinjuku, Tokyo, Japan) supplied with a Color View II camera (Soft Imaging System, Münster, Germany). For electron microscopy, samples were sectioned with a diamond knife using an ultramicrotome LKB-III (LKB, Sweden), placed on formvar-coated grids and stained with uranyl acetate and lead citrate solutions. Then, thin sections were analyzed and photographed using an electron microscope H-500 (Hitachi, Ibaraki, Japan).
9
Ultrastructural Analysis of Ovules
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For electron microscopy, the same preparations of ovules embedded in an epoxy-araldite composition were used. Ultrathin sections were made using a 1.5 mm wide diamond knife on an LKB-V ultramicrotome (LKB, Bromma, Sweden). Sections were placed on mixtures or grids coated with formvar and contrasted with solutions of uranyl acetate and lead citrate according to the standard method. Thin sections were then analyzed and photographed using an H-500 (Hitachi, Ibaraki, Japan) and JEM-1400 (Jeol, Akishima, Tokyo, Japan) electron microscope.
10
Ultrastructural Analysis of Plant Cells by TEM
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For transmission electron microscopy (TEM), epidermal strips with a circumferential length of ~4 mm and a width of 2 mm were peeled in the circumferential direction from the same hypocotyl, coleoptile, or peduncle regions as the strips that were used for Nomarski microscopy. A solution of 300 mM mannitol in 50 mM phosphate buffer (pH 7) was applied for 15 min to plasmolyse the samples. The samples were then immersed in fixative solution (2.5% glutaraldehyde, 2% caffeine) for 3 h, buffered in 300 mM mannitol in 50 mM phosphate buffer (pH 7) post-fixed with 1% OsO4 for 2 h, rinsed in water, and dehydrated in a graded ethanol series. After dehydration, the samples were embedded in Epon resin. Ultrathin longitudinal–radial sections (the plane across the periclinal walls and parallel to the long cell axis), 90 nm thick, were mounted on copper grids (200 mesh), double-stained with uranyl acetate and lead citrate, and examined by TEM (Hitachi H500).
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