The inner mesocarp was comminuted with a scalpel into small cubes (1mm3) which were dipped in 0.05M Sorensen buffer (pH 7.3) containing 2.5% glutaraldehyde and gently stirred for 16h at 4 °C. The cubes were then rapidly rinsed with distilled water (3×10min), then post-fixed in 1% aqueous osmium tetroxide containing 3% sucrose for 2h at 20 °C in the dark. They were then dehydrated in an ethanol series (30, 50, 70, and 90%; 10min each) and finally for 15min in ethanol; they were then embedded in Epon EmBed 812 using an Automated Microwave Tissue Processor (Leica EM AMW). Ultrathin sections (thickness 80nm) were obtained with a Leica-Reichert Ultracut E ultramicrotome, then stained with uranyl acetate in ethanol. Sections were then mounted on Ni-grids and examined with a Hitachi 7100 electron microscope.
7100 electron microscope
Manufactured by Hitachi
Sourced in Japan
The Hitachi 7100 electron microscope is a high-performance instrument designed for advanced materials analysis. It utilizes an electron beam to produce detailed images and data about the structure and composition of samples at the nanoscale level. The 7100 model offers high resolution, stability, and versatility for a wide range of applications in scientific research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Em amw, by Leica (2 mentions)
Veleta ccd camera, by Olympus (2 mentions)
Ultrostain 2, by Leica (2 mentions)
Em uc6 ultramicrotome, by Leica (2 mentions)
Megapixel side mounted tem ccd camera, by Olympus (2 mentions)
Mega view 2, by Olympus (2 mentions)
Embed 812, by Leica (1 mentions)
H7500 tem, by Hitachi (1 mentions)
Mega view 3 camera, by Olympus (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
14 protocols using 7100 electron microscope
1
Ultrastructural Analysis of Mesocarp
2
Electron Microscopy of Plant Cell Ultrastructure
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The samples were prepared and analyzed as described previously (Verdeil et al., 2001 (link)). Samples taken from the central cell layers in the center of the primary AZ between vascular strand parenchyma cells (1 mm3 cubes of tissue) were fixed in a 0.05 M Sorensen buffer solution (pH 7.3) containing 2.5% glutaraldehyde and gently stirred for 16 h at 4°C. Cubes were then rapidly rinsed with distilled water (3 × 10 min), then post-fixed in 1% aqueous osmium tetroxide containing 3% sucrose for 2 h at 20 °C in the dark. They were then dehydrated in an EtOH dilution series (30, 50, 70, and 90% EtOH; 10 min each) and finally for 15 min in pure EtOH; samples were then embedded in Epon EmBed 812 using an Automated Microwave Tissue Processor (Leica EM AMW). Ultrathin sections (thickness around 80 nm) were obtained with a Leica-Reichert Ultracut E ultramicrotome, and then stained with uranyl acetate in EtOH. Sections were then mounted on Ni-grids and examined with a Hitachi 7100 electron microscope.
3
Ultrastructural Analysis of Spinal Cord
4
Ultrastructural Analysis of Dorsal Horn
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For electron microscopy, after development of the immunoperoxidase reaction, sections were first treated with 1% OsO4 in 0.1 m PB for 10 min in the dark, on ice, and then dehydrated in an ascending series of ethanol solutions, followed by acetonitrile. An additional treatment with uranyl acetate (1% in 70% ethanol for 10 min in the dark, on ice) was included during the dehydration process. Sections were embedded in Durcupan (ACM, Fluka, Buchs, Switzerland). Areas of interest containing the dorsolateral fasciculus (Lissauer’s tract) and the superficial laminae were cut from the dorsal horn of lumbar segments of both MGL+/+ and MGL−/− spinal cords, and re-sectioned to produce ultrathin 50-nm thin sections with a Leica EM UC6 Ultramicrotome (Leica Microsystems). These sections were collected on a Formvar-coated single-slot copper grid, contrasted with lead citrate (Ultrostain2; Leica), and examined with a Hitachi 7100 electron microscope (Hitachi High-Technologies, Tokyo, Japan). Electron micrographs at 40 000 × magnification were acquired with a Veleta CCD camera (Olympus Soft Imaging Solutions, Munster, Germany).
5
Transmission Electron Microscopy of EVs
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For TEM analysis, EVs were generated and isolated from NSCs following the protocol described above [10 (link)]. Following the last ultracentrifugation step, the supernatant was carefully removed, and the pellets were fixed with 4% PFA-PBS for 10 minutes at RT, and then kept overnight at 4°C. After removal of the fixative and a short rinse with PBS, the pellets were postfixed in 1% OsO4 (Taab, Aldermaston, Berks, UK) for 30 minutes, rinsed with distilled water, dehydrated in graded ethanol, including block staining with 1% uranyl acetate in 50% ethanol for 30 minutes, and embedded in Taab 812 (Taab). Overnight polymerisation at 60°C was followed by sectioning, and the ultrathin sections were analysed using a Hitachi 7100 electron microscope (Hitachi, Chiyoda City, Tokyo, Japan) equipped with Veleta, a 2,000 × 2,000 MegaPixel side mounted TEM CCD camera (Olympus, Shinjuku City, Tokyo, Japan).
6
Transmission Electron Microscopy of Muscle Mitochondria
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In the HSP72Tg study, standard processing methods were used. The muscle was fixed in 2.5% glutaraldehyde, osmicated with 2.5% osmium tetroxide, dehydrated through a graded series of acetone solutions, and embedded in epon-araldite. Ultra-thin sections were cut on a Reichert-Jung Ultra-S microtome and collected on Nickel Grids. The sections were stained with uranyl acetate and lead citrate and viewed on a Hitachi H7500 TEM. Random images were taken at a magnification of 20,000. The area of the field of view was calculated, and mitochondria were counted and expressed as number of mitochondria per micrometers squared. Ten different areas of muscle were calculated for each animal and averaged. For the BGP-15 studies, sections were prepared in a similar manner to what is described above; however, they were imaged in a different location on a Hitachi 7100 electron microscope, and mitochondrial areas were calculated by ImageTool (University of Texas Health Science Center, San Antonio, TX).
7
Characterization of EV Morphology and Size
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In order to characterize the morphology and size of the different EV fractions, EV pellets were fixed with 4% paraformaldehyde in PBS for at least 60 min at room temperature and analyzed by transmission electron microscopy (TEM). After washing with PBS, the preparations were postfixed in 1% osmium tetroxide (OsO4, Taab, Aldermaston, Berks, UK). This was followed by rinsing with distilled water. The pellets were dehydrated in graded ethanol including block staining with 1% uranyl-acetate in 50% ethanol for 30 min, and were embedded in Taab 812 (Taab). An overnight polymerization of samples at 60 °C was followed by sectioning, and the ultrathin sections were analyzed using a Hitachi 7100 electron microscope (Hitachi Ltd., Japan) equipped by Veleta, a 2000 × 2000 MegaPixel side-mounted TEM CCD camera (Olympus).
8
Electron Microscopy of Extracellular Vesicles
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In order to characterize the morphology and size of the different EV preparations, pellets were fixed with 4% paraformaldehyde in 0.01M PBS for 60 min at room temperature. Following washing with PBS, the preparations were postfixed in 1% OsO4 (Taab, Aldermaston, Berks, UK) for 30 min. After rinsing the intact fixed pellets within the centrifugation tubes with distilled water, the pellets were dehydrated in graded ethanol, including block staining with 1% uranyl-acetate in 50% ethanol for 30 min, and were embedded in Taab 812 (Taab). Overnight polymerization of samples at 60°C was followed by sectioning, and the ultrathin sections were analyzed using a Hitachi 7100 electron microscope (Hitachi Ltd., Japan) equipped with a Megaview II (lower resolution, Soft Imaging System, Germany) digital camera.
9
Ultrastructural Analysis of Wheat Chloroplasts
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Seedling leaves of two synthetic hexaploid wheat lines, Ldn/PI476874 for WT and Ldn/KU-2111 for severe chlorosis, grown at 23°C for 3 weeks, were used for TEM. The leaf blades were cut into 1 mm2 pieces and then incubated in a freshly prepared solution of 5 mM CeCl3 buffered with 50 mM 3-(N-morpholino)propanesulfonic acid at pH 7.2 for 1 h. The fixation of samples and the sectioning of ultrathin sections 90 nm thick were conducted according to our previous report [20 (link)]. Sections were examined with a Hitachi-7100 electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV. Three sections cut from leaf resin blocks of two plants were used for ultrastructural analysis of chloroplasts. More than 25 mesophyll cells were examined in each of the sections. The areas of three different structures, such as chloroplasts, grana and plastoglobuli, were measured by Image J software (National Institutes of Health, USA; http://imagej.nih.gov/ij/ ).
10
Leaf Ultrastructure Analysis Protocol
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Leaf pieces were fixed in 2.5% (w/V) glutaraldehyde in 70 mM Na-K phosphate (pH 7.2) buffer for 2 h in the dark as in Ounoki et al. (2021) (link). Post-fixation was performed in 1% (w/V) OsO4 for 3 h, in the same buffer. After dehydration in an alcohol series, the samples were embedded in Durcupan ACM (Fluka); 60-nm ultrathin sections were cut with Reichert Jung Ultracut E microtome (Reichert-Jung AG, Vienna, Austria). The sections were stained with uranyl acetate and lead citrate. Ultrathin sections were examined in a Hitachi 7100 electron microscope (Hitachi Ltd., Tokyo, Japan) at 75 kV accelerating voltage. TEM micrographs were taken with a MegaView III camera (Soft Imaging System, Münster, Germany).
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