Samples were fixed as described above, substituting phosphate buffer for 0.1 M sodium cacodylate buffer (Agar Scientific AGR1103) and embedded in epoxy resin (Agar Scientific AGR1031). Ultra-thin (50 nm) sections were cut using a Leica EM UC7 ultratome with a diamond knife. The sections were mounted on Pioloform-coated copper grids, stained 30 min in 2% uranyl acetate and 4 min in Reynolds’ lead citrate [78 (link)] and examined at 100 kV using a JEOL JEM-1400 Plus transmission electron microscope. Micrographs were recorded with a JEOL Matataki CMOS camera using the TEM Centre for JEM1400 Plus software.
Em uc7 ultratome
Manufactured by Leica camera
Sourced in Germany, Japan
The EM UC7 ultratome is a high-performance microtome designed for the preparation of ultra-thin sections for electron microscopy. It features precise and reliable sectioning, ensuring the production of consistent and uniform samples for analysis.
Automatically generated - may contain errors
Lab products found in correlation
H 7650 tem, by Hitachi (10 mentions)
Model h 7650 tem, by Hitachi (8 mentions)
Model h 7650, by Hitachi (5 mentions)
H 7650, by Hitachi (4 mentions)
E 1010, by Hitachi (3 mentions)
Hcp 2, by Hitachi (2 mentions)
Su8010 sem, by Hitachi (2 mentions)
H 7650 transmission electron microscope, by Hitachi (2 mentions)
Model hcp 2, by Hitachi (2 mentions)
Agr1031, by Agar Scientific (1 mentions)
Jem 1400plus, by JEOL (1 mentions)
Dm2500 microscope, by Leica camera (1 mentions)
Su8010 scanning electron microscope, by Hitachi (1 mentions)
E 1010 ion sputter, by Hitachi (1 mentions)
Hcp 2 critical point dryer, by Hitachi (1 mentions)
H 7500 transmission electron microscope, by Hitachi (1 mentions)
Su3500 scanning electron microscope, by Hitachi (1 mentions)
H 7650 electron microscope, by Hitachi (1 mentions)
Spurr resin, by Merck Group (1 mentions)
Model jem 1230, by JEOL (1 mentions)
35 protocols using em uc7 ultratome
1
Transmission Electron Microscopy Protocol
2
Ultrastructural Analysis of Biological Samples
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For transmission electron microscopy samples were fixed overnight at 4 °C in 2.5% glutaraldehyde in phosphate buffer with addition of Sucrose (pH 7.4; 750 mOsM), and postfixed in 1% OsO4 in the same buffer (an hour at room temperature). After washing with the same buffer, the specimens were dehydrated through an ethanol series and acetone and embedded in Epon-812 embedding media (Fluka). Semithin (1 μm thick) and ultra-thin (60–80 nm) sections were cut with a Leica EM UC7 ultratome. Semi-thin sections were stained with methylene blue and studied under Leica DM2500 microscope. Ultra-thin sections were stained with uranyl acetate followed by Reynolds lead nitrate46 (link) and examined under transmission electron microscope JEOL JEM 1400 in Resource Centres “Chromas” and “Molecular and Cell Technologies” of the Research Park of St Petersburg State University. Specimens for SEM were dehydrated in ethanol series and acetone, critical point-dried in Hitachi critical point dryer HCP- 2, mounted on stubs, coated with platinum using Giko IB-5 Ion coater, and viewed under FEI Quanta 250 scanning electron microscope in “Taxon” Research Resource Center (http://www.ckp-rf.ru/ckp/3038/ ) of the Zoological Institute of the Russian Academy of Sciences.
3
Ultrastructural Analysis of Plant Tissues
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Small segments from the middle part of the leaves were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.0) by vacuum infiltration, and treated for more than 4 h. After washing in buffer, the samples were postfixed in buffered 1% (w/v) osmium tetroxide, washed, dehydrated in an ethanol series, transferred to absolute acetone, and embedded in Spurr resin. Specimen was placed in eppendorf contained resin and heated at 70°C for more than 9 h. The specimen was sectioned in LEICA EM UC7 ultratome and the sections were stained with 2% (w/v) uranyl acetate and then 3% (w/v) alkaline lead citrate for 5–10 min, respectively. The samples were observed in transmission electron microscopy (Model H7650; Hitachi; Japan) at 75 kV.
4
Ultrastructural Analysis of Leaf Epidermal Cells
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The upper second leaves of the WT and es5 plants were collected at tillering stage and cut into small pieces. The samples were fixed with 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.0) for more than 4 h at 4 °C. The samples were washed three times in phosphate buffer and post fixed with 1% OsO4 in phosphate buffer for 1–2 h. Then the samples were washed 3 times with phosphate buffer and dehydrated by a graded series of ethanol (30 to 100%) for 15–20 min at each step. Finally, the samples were transferred to absolute acetone for 20 min. The samples were placed in a series of resin spur mixtures overnight. Specimens were placed in Eppendorf microtubes containing spur resin and heated at 70 °C for more than 9 h. Specimens were sectioned in Leica EM UC7 ultratome and sections were stained with uranyl acetate and alkaline lead citrate for 5 and 10 min, respectively, and observed by TEM (Hitachi Model H-7650).
5
Electron Microscopy of H9-CMs
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H9‐CMs were dissociated with Tripsin‐EDTA, scrapped into a 1.5‐mL microcentrifuge tube and centrifuged and then fixed with cold 2.5%‐glutaraldehyde in 0.1 mol/L phosphate buffer overnight at 4°C. The specimen was postfixed with 1% OsO4 in phosphate buffer and dehydrated by a graded series of ethyl‐alcohol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15‐20 minutes at each step and then transferred to absolute acetone for 20 minutes. The specimen was placed in 1:1 mixture of absolute acetone and final spur resin mixture for 1 hour at room temperature, and transferred to 1:3 mixture of absolute acetone and final spur resin mixture for 3 hours, and then transferred to final spur resin mixture overnight. The specimen was placed in 1.5‐mL tube contained spur resin, heated at 70°C for more than 9 hours and sectioned using a LEICA EM UC7 ultratome. The sections were then stained with uranyl acetate and alkaline lead citrate for 5‐10 minutes. Pictures were observed using a transmission electron microscopy (Hitachi, Model H‐7650).
6
Ultrastructural Analysis of Mitochondria and Cilia
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Mitochondria morphology was visualized with transmission electron microscopy. Cells were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in a graded series of ethanol solution, washed in acetone, and embedded in resin mixture. Sections were obtained by EM UC7 ultratome (Leica, Germany), and then stained by uranyl acetate and lead citrate. Images were taken by H-7650 transmission electron microscope (Hitachi, Japan). The ultrastructure of cilium in cells was observed using scanning electron microscopy. After dehydrated through an ethanol series and washed with pure ethanol, cell samples were inserted into HCP-2 critical point dryer (Hitachi, Japan) until dry, coated with gold-palladium in E−1010 ion sputter (Hitachi, Japan) for 4–5 min and observed in the SU-8010 scanning electron microscope (Hitachi, Japan).
7
Microscopic Characterization of L. monocytogenes
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The L. monocytogenes strains were grown to exponential phase (OD600nm = 0.8), followed by nisin (800 μg/mL) treatment in experimental groups for 1 h, after which the bacteria cultures were spun down and fixed with 2.5% glutaraldehyde in PBS for more than 4 h. The samples were washed three times in PBS, postfixed with 1% OsO4 for 1 h, and washed three times. Then the dehydration of samples used a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%). The samples were dehydrated by alcohol for 20 min, and dehydrated in Hitachi Model HCP-2 critical point dryer. Later we can coat with gold-palladium in Hitachi Model E-1010 ion sputter for 4 to 5 min and observe in Hitachi Model SU-8010 SEM. The samples were transferred to absolute acetone for dehydration, placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture, then transferred to 1:3 mixture of absolute acetone and the final resin mixture and to the final Spurr resin mixture for overnight. Specimens were heated at 70°C, then sectioned in LEICA EM UC7 ultratome, and sections were stained by uranyl acetate and alkaline lead citrate for 5 to 10 min respectively and observed in Hitachi Model H-7650 TEM.
8
TEM and SEM Imaging of Leaf Ultrastructure
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For TEM, the flag leaves of nlg1 and YD32 were collected and fixed in 2.5% glutaraldehyde fixative solution for 2 days. The fixed samples were washed with phosphate buffer saline 3 times and then post-fixed in 1% OsO4 solution for 1 h, followed with a uranyl acetate staining, gradient ethanol dehydration, embedded in Spurr and sliced with Leica EM UC7 ultratome. Finally, the 70 nm sectioning samples were stained again and observed under a Hitachi H-7500 Transmission Electron Microscope. For SEM, the fresh samples were observed with a Hitachi SU3500 Scanning Electron Microscope.
9
TEM Analysis of Mitochondrial Morphology in PCV2-Infected Cells
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Transmission electron microscopy (TEM) was used to assess the mitochondrial morphology. PK-15 cells were incubated in a six-well plate. Mock- and PCV2-infected cells (48 hpi) were prepared for the TEM. The specimens were first fixed with 2.5% glutaraldehyde in a phosphate buffer (PB) (0.1 M, pH 7.0) for 4–5 h, and then washed three times with PB for 15 min at each step. The specimens were postfixed with 1% OsO4 in PB for 1–2 h and washed three times with PB as per above. Dehydration of the specimens was conducted using a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for about 15 to 20 min at each step, and then using absolute acetone for 20 min. For embedding and ultrathin sectioning, specimens were placed in Eppendorf tubes containing Spurr resin and heated at 70 °C for at least 9 h. The LEICA EM UC7 ultratome (Wetzlar, Germany) was used to prepare the sections, which were stained with uranyl acetate and alkaline lead citrate for 5 to 10 min, and then observed using the Hitachi Model H-7650 TEM (Tokyo, Japan).
10
Ultrastructural Analysis of Mitochondria
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Cell samples were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide. Samples were then dehydrated in acetones, embedded in Spurr resin. Ultra-thin sections were cut and stained with uranyl acetate and lead citrate. The sample block was then sectioned with a LEICA EM UC7 ultratome. Sections were collected on copper grids for imaging under a Hitachi H-7650 electron microscope. Ultrastructural of the mitochondrial were recorded by a transmission electron microscope (Hitachi H-7650, Tokoy, Japan).
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