Quetol 651 resin

Manufactured by Nisshin EM

Sourced in Japan

Quetol-651 is a low-viscosity epoxy resin for electron microscopy specimen preparation. It is designed for embedding biological samples prior to ultrathin sectioning and observation.

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Lab products found in correlation

Bz 9000 microscope, by Keyence (1 mentions) Ultracut uct ultramicrotome, by Leica (1 mentions) H 7650, by Hitachi (1 mentions) Jem 1210 transmission electron microscope, by JEOL (1 mentions) Lead citrate, by Merck Group (1 mentions) Tecnai g2 20 electron microscope, by Thermo Fisher Scientific (1 mentions) Ultracut s, by Leica (1 mentions) Em pact2, by Leica (1 mentions) Lead staining solution, by Merck Group (1 mentions) Uranyl acetate, by Merck Group (1 mentions) Jem 1400 plus electron microscope, by JEOL (1 mentions) Em 14830ruby2, by JEOL (1 mentions)

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Quetol 651 (19 citations) Quetol 651 epoxy resin (2 citations)

7 protocols using quetol 651 resin

Microscopic Analysis of Nicotiana-Fern Graft

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Example 10

The tests of Examples 10 and 11 were performed on grafting between a Nicotiana benthamiana scion and a fern (Cyrtomium fortunei) stock at the first month after grafting. The graft site was cut, and a traverse free-hand section with a thickness of several hundreds of μm was produced under a stereoscopic microscope while immersing the sample in a fixing solution (2% paraformaldehyde, 2% glutaraldehyde, 0.05 M cacodylate buffer, pH 7.4). After degassing was repeated several times in a fixing solution with the same composition, fixation was performed at 4° C. overnight. On the next day, washing with a 0.05 M cacodylate buffer was performed 3 times for 30 minutes per time. Then, fixation was performed in another fixing solution (2% osmium tetroxide, 0.05 M cacodylate buffer, pH 7.4) at 4° C. for 3 hours. The sample was dehydrated with ethanol, and then embedded in Quetol-651 resin (Nisshin EM). A section with a thickness of 150 μm was produced by a microtome, stained with a 0.5% toluidine blue aqueous solution, and observed and photographed by an optical microscope. FIG. 11 shows the results.

As shown in FIG. 11, it was observed that the tissue of the Nicotiana plant was fused with the vascular bundle of the fern.

US11129337B2. Grafted plant body and method for producing same (2021-09-28). National University Corporation Nagoya University [JP]. Inventors: Michitaka Notaguchi [JP].

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Plant Anther Fixation and Sectioning

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Anther samples were fixed with 4% glutaraldehyde in 60 mM Hepes (pH 7.0) containing 0.125 M sucrose. After dehydration in a graded series of ethanol/water mixtures, the samples were embedded in Quetol 651 resin (Nisshin EM) with formulation for plant material (Ellis, 2016 (link)). Semi-thin (2 μm) transverse sections were prepared from at least eight resin blocks per sample and stained with 0.2% toluidine blue. Stained sections were examined using a BZ-9000 microscope (Keyence). Histochemical GUS staining of IRE1C promoter–GUS plants was performed as previously described (Iwata et al, 2008 (link)).

Mishiba K.I., Iwata Y., Mochizuki T., Matsumura A., Nishioka N., Hirata R, & Koizumi N. (2019). Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis. Life Science Alliance, 2(5), e201900459.

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Transmission Electron Microscopy Sample Preparation

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Tissues were fixed in a solution of 4% paraformaldehyde and 0.5% glutaraldehyde/0.1 M cacodylate. Osmium tetroxide fixation was performed to stabilize lipids following paraformaldehyde/glutaraldehyde fixation. After fixation, samples were dehydrated in increasing concentrations of ethanol. Then dehydrated samples were embedded in Quetol651 resin (Nisshin EM), followed by polymerization at 60°C. The 80 nm ultrathin sections were prepared using a diamond knife (ULTRA, 2.5 mm width, angle 45°, Nisshin EM) equipped with ultracut UCT ultramicrotome (Leica Microsystems) and then mounted on nickel grids (EM fine grid F100, 219 μm 263-N, Nisshin EM). The sections were stained with uranyl acetate, followed by lead citrate to enhance contrast. These processes were done by dipping the grids in droplets of stain, followed by water rinses. After washing with pure water, the grids were imaged with a transmission electron microscope (1200EX; JEOL Ltd. or H7650; Hitachi High-Technologies Corp.) by using the transmission electron image mode and an accelerating voltage of 80 kV.

Park G., Fukasawa K., Horie T., Masuo Y., Inaba Y., Tatsuno T., Yamada T., Tokumura K., Iwahashi S., Iezaki T., Kaneda K., Kato Y., Ishigaki Y., Mieda M., Tanaka T., Ogawa K., Ochi H., Sato S., Shi Y.B., Inoue H., Lee H, & Hinoi E. (2023). l-Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis. JCI Insight, 8(7), e154925.

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Transmission Electron Microscopy of Bacterial Cell Morphology

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To examine bacterial cell shape, the cells were washed and negatively stained on carbon-coated grids with 1 % ammonium molybdate. To prepare ultrathin sections, the cells were fixed with 4 % paraformaldehyde and 5 % glutaraldehyde in 30 mM HEPES buffer (pH 7.4) overnight at 4 °C. The samples were post-fixed with 1 % osmium tetroxide for 2 h and then with 0.5 % uranyl acetate for 30 min. The fixed cells were dehydrated in a series of 25–100 % ethanol and embedded in Quetol-651 resin (Nisshin EM). The ultrathin sections were stained with 1 % uranyl acetate and 1 % lead citrate. The stained samples (bacterial cells and ultrathin sections) were observed using a JEM-1210 transmission electron microscope (JEOL).

Narita Y., Sato K., Yukitake H., Shoji M., Nakane D., Nagano K., Yoshimura F., Naito M, & Nakayama K. (2014). Lack of a surface layer in Tannerella forsythia mutants deficient in the type IX secretion system. Microbiology, 160(Pt 10), 2295-2303.

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High-Pressure Freezing and Electron Microscopy

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A cell pellet was harvested from approx. 300 ml of two-week-old culture by centrifuging at 1,200 x g for 4 min, and then was high-pressure frozen (at -196 °C under 2,181 bar) using a Leica EM PACT2 (Leica). The frozen pellet was transferred to 1% osmium tetroxide in HPLC grade acetone and substituted for 118 hours at -85 °C, then warmed to -20 °C over five hours. After this, the specimen was warmed to 4 °C over 48 hours and then washed in acetone. Subsequently, the specimen was transferred through increasing concentrations of Quetol 651 resin (Nisshin EM Co., Ltd.) in acetone: 1:2 resin/acetone for 1 hr, 1:1 for 2 hr, and 2:1 for 3 hr. The specimen was then embedded in pure Quetol 651 resin overnight at room temperature, and this step repeated again with fresh resin. The embedded specimen was polymerized overnight in fresh resin at 65 °C.
Ultrathin sections (ca. 70 nm) of both EM blocks were prepared using an ultramicrotome (Ultracut S; Leica), double stained with 2.0% uranyl acetate and 2.0% lead citrate (Sigma-Aldrich Co.), and observed in a Tecnai G2 20 electron microscope (FEI).

Yabuki A., Gyaltshen Y., Heiss A.A., Fujikura K, & Kim E. (2018). Ophirina amphinema n. gen., n. sp., a New Deeply Branching Discobid with Phylogenetic Affinity to Jakobids. Scientific Reports, 8, 16219.

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Histological Analysis of Graft Vessel Development

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Example 7

Histological Observation of Vessel Element

The graft site was cut, and a traverse free-hand section with a thickness of several hundreds of μm was produced under a stereoscopic microscope while immersing the sample in a fixing solution (2% paraformaldehyde, 2% glutaraldehyde, 0.05 M cacodylate buffer, pH 7.4). After degassing was repeated several times in a fixing solution with the same composition, fixation was performed at 4° C. overnight. On the next day, washing with a 0.05 M cacodylate buffer was performed 3 times for 30 minutes per time. Then, fixation was performed in another fixing solution (2% osmium tetroxide, 0.05 M cacodylate buffer, pH 7.4) at 4° C. for 3 hours. The sample was dehydrated with ethanol, and then embedded in Quetol-651 resin (Nisshin EM). A section with a thickness of 150 μm was produced by a microtome, stained with a 0.5% toluidine blue aqueous solution, and observed and photographed by an optical microscope. FIG. 8 shows the results. A shows a schematic diagram of the graft site, and the dotted line shows the observed area. B shows the observation results of the longitudinal section of the graft site, and C and D show the magnified observation results of the dotted-line areas in B.

As shown in FIG. 8, it was revealed that vessel elements were formed in random directions in the parenchyma developed in the scion.

US11129337B2. Grafted plant body and method for producing same (2021-09-28). National University Corporation Nagoya University [JP]. Inventors: Michitaka Notaguchi [JP].

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Transmission Electron Microscopy of Arabidopsis Leaves

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TEM was performed by Tokai Electron Microscopy Inc. (Nagoya, Japan), as described previously (Itoh et al., 2018 (link)). Briefly, primary leaves of 10-day-old Col, FL4-4, parc6-1, and parc6-5/suba2 seedlings were sampled from 10:30 to 12:00 and fixed in 2% paraformaldehyde and 2% formaldehyde in 0.05 M cacodylate buffer (pH 7.4) at 4°C. The fixed leaf samples were washed with 0.05 M cacodylate buffer, postfixed with 2% osmium tetroxide in 0.05 M cacodylate buffer, and dehydrated in a graded ethanol series (50, 70, 90, and 100%). Samples were then embedded in a 70:30 mixture of propylene oxide and Quetol-651 resin (Nisshin EM, Tokyo, Japan). Ultrathin (80 nm thick) sections were prepared using a diamond knife and then stained with 2% uranyl acetate and lead staining solution (Sigma-Aldrich, Tokyo, Japan). Grids were observed using a JEM-1400Plus electron microscope (JEOL, Tokyo, Japan) equipped with a CCD camera (model EM-14830RUBY2, JEOL).

Ishikawa H., Yasuzawa M., Koike N., Sanjaya A., Moriyama S., Nishizawa A., Matsuoka K., Sasaki S., Kazama Y., Hayashi Y., Abe T., Fujiwara M.T, & Itoh R.D. (2020). Arabidopsis PARC6 Is Critical for Plastid Morphogenesis in Pavement, Trichome, and Guard Cells in Leaf Epidermis. Frontiers in Plant Science, 10, 1665.

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