To examine the size and structure of the purified NPs, microscopic evaluations using TEM and cryo-EM were performed. For TEM analysis, a drop of the NPs was placed onto a formvar/carbon-coated TEM grid (SPL). The grid was negatively stained with 2% uranyl acetate, dried, and examined using a JEM-1011 electron microscope (JEOL) at an accelerating voltage of 80 kV. The particle sizes were calculated using Camera-Megaview III (Soft imaging system-Germany) for measuring the NPs in random image fields. For cryo-EM, the NPs were placed onto plasma-treated formvar/carbon 200 copper grid (EMS) and negatively stained with 2% uranyl acetate. The grid was accelerated at 200 kV with an FEI CryoTecnai F20 cryo-EM microscope made available through the Korean Institute of Science and Technology. The NPs were examined and photographed in high resolution.
Mega view 3 camera
Manufactured by Olympus
Sourced in Germany, Japan, United States
The Mega View III camera is a high-performance microscope camera designed for advanced imaging applications. It features a large, high-resolution sensor and advanced optics to capture detailed, clear images. The camera is compatible with a variety of microscope systems and can be used for various imaging tasks.
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Lab products found in correlation
Beem capsules, by Electron Microscopy Sciences (6 mentions)
Item 5, by Olympus (6 mentions)
1010 electron microscope, by JEOL (5 mentions)
Spurr resin, by Electron Microscopy Sciences (5 mentions)
Cm120 electron microscope, by Philips (4 mentions)
Ultracut uct, by Leica camera (4 mentions)
Item software, by Olympus (3 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Leica dmlb, by Leica Microsystems (3 mentions)
Jem 1011, by JEOL (2 mentions)
Em uc6 ultramicrotome, by Leica camera (2 mentions)
Poly bed 812, by Polysciences (2 mentions)
Jum 7 ultramicrotome, by JEOL (2 mentions)
Microtome, by JEOL (2 mentions)
Jem 1010 transmission electron microscope, by JEOL (2 mentions)
Embed 812, by Electron Microscopy Sciences (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Cm12 transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
Epon 812, by Merck Group (2 mentions)
63 protocols using mega view 3 camera
1
Microscopic Evaluation of Purified NPs
2
Exosome Visualization via Transmission Electron Microscopy
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A Formvar-carbon-coated electron microscope grid was placed with the formvar side down on top of an exosome drop for approximately 1 min. The grid was removed, blotted with filter paper, and placed onto a drop of 2% uranyl acetate for 15 s. The excess uranyl acetate was removed, and the electron microscope grid was examined and photographed for transmission electron microscopy (TEM). All thin sections were observed under a transmission electron microscope (JEM-1011; JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV. Images were captured with a side-mounted Camera-Megaview III (Soft Imaging System, Münster, Germany) [24 (link)].
3
Ovary Grafts Ultrastructural Analysis
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Ovary grafts collected at 2 weeks post KCT surgery were processed for transmission electron microscopy by the University of Wisconsin Electron Microscopy Service. Briefly, samples were immersion fixed for 2 hours in 2.5% glutaraldehyde, 2% PFA buffered in 0.1M sodium phosphate buffer (PB) at room temperature (RT). After rinsing, samples were post-fixed in 1% osmium tetroxide, 1% potassium ferrocyanide in 0.1M PB for 1 hour at RT, rinsed, and then stained in saturated aqueous uranyl acetate for 2 hours at RT. Dehydration was performed at RT in a graded ethanol series and then transitioned in propylene oxide (PO). Fully dehydrated samples were then infiltrated in increasing concentrations of PolyBed 812 (Polysciences Inc. Warrington, PA) and PO mixtures. Embedding and polymerization took place in fresh PolyBed 812 for 48 hours at 60°C. Semi-thin sections (1 mm) were first stained with methylene blue/Azure II for light microscopic inspection. The samples were then sectioned on a Leica EM UC6 ultramicrotome at 90nm. The sections were collected on Cu, 300 mesh thin-bar (EMS Hatfield, PA), and post-stained in uranyl acetate and lead citrate. The sectioned samples were viewed at 80kV on a Philips CM120 transmission electron microscope, equipped with MegaView III camera (Olympus Soft Imaging System Lakewood, CO).
4
TEM Imaging of Insect Hemocytes
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For TEM assays, hemolymph collected from 10 insects was pooled and centrifuged at 500× g for 10 min at room temperature [31 (link),32 (link)] to enrich the preparation with hemocytes without inducing visible cell damage. The supernatant was discarded and the pellet was suspended and fixed in Karnovsky mixture containing 4% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer for 2 h and processed as previously described [33 (link)]. Fixed cells were washed and treated with 1% OsO4, dehydrated in a series of acetone solutions, and embedded in Embed 812 resin. After a 48-h polymerization stage, 200 nm semithin sections obtained using a Jeol microtome (Tokyo, Japan) were stained with toluidine blue for high-resolution light microscopy analysis. Finally, 90-nm ultrathin sections obtained in a JUM-7 ultramicrotome (Jeol, Tokyo, Japan) were placed on grids, post-stained with uranyl acetate/lead citrate solutions, examined in an electron microscope (Zeiss Leo 906-E, Oberkochen, Germany), and photographed with a Megaview III camera (Olympus, Center Valley, PA, USA).
5
Ultrastructural Analysis of Artery Samples
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Ultrastructural analysis was performed on artery samples fixed in 2.5% glutaraldehyde in 0.1 mmol/L cacodylate buffer (pH 7.4) at 4°C. Then fragments were post-fixed in 1% osmium tetroxide, dehydrated using graded alcohol series, and embedded in epoxy resin. Semi-fine sections (0.5 μm) were stained using toluidine blue. Ultrastructure sections (60 nm) were contrast-enhanced using uranyl acetate and lead citrate, and examined using a JEOL 1010 electron microscope (JEOL, Ltd., Tokyo, Japan) with a MegaView III camera (Olympus Soft Imaging Systems GmbH, Münster, Germany).
6
High-Pressure Freezing for TEM
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Transmission electron microscopy samples were prepared by high pressure freezing technique (HPF), a very rapid method that prevents the formation of ice crystals that could damage the cells ultrastructure. Briefly, 5 × 108P. papillatum cells were concentrated by centrifugation and processed as described previously [61 (link)]. Ultrathin sections were cut using an ultramicrotome (Leica Microsystems) and collected on copper grids, which were contrasted in ethanolic uranyl acetate and lead citrate and observed using a JEOL 1010 microscope at accelerating voltage of 80 kV. Images were captured with an Olympus Mega View III camera.
7
Transmission Electron Microscopy of Bacteria
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To obtain TEM images, Formvar‐coated copper grids were floated on a drop (5–10 μL) of appropriately diluted bacteria for 1 min. Then, the grids plus absorbed bacteria were rinsed rapidly with distilled water and floated on a solution of uranyl (2%) for 30 s. Then, the grids were removed with forceps, rinsed with distilled water and the excess liquid was drained off with the edge of a filter paper and preparations were air dried for 5 min. Finally, the specimens were examined with a Zeiss LEO906 TEM (Carl Zeiss, Oberkochen, Germany) (operated at an accelerating voltage of 100 kV) and photographed with a Megaview III camera (Olympus, Center Valley, PA, USA).
8
Ultrastructural Analysis of Cell Lines
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HCT-116 and RKO (control and treatment) cell lines were centrifuged and the culture medium above the pellets was replaced with 2.7% glutaraldehyde (Electron Microscopy Sciences, Hatfield, USA) in 0.1 M phosphate buffer, pH 7.4. Prefixation was performed at 4 °C for 2 h. The pellets were washed four times with 0.1 M phosphate buffer, and then postfixed for 24 h at 4 °C with 1.5% OsO4 (Sigma-Aldrich) in 0.15 M phosphate buffer, pH 7.4. The cells were embedded in EMBed-812 (Electron Microscopy Sciences, Hatfield, USA), after a previous dehydration in an acetone series. Polymerization of the resin was performed at 60 °C for 72 h. Ultrathin sections were cut with a DiATOME diamond knife (DiATOME, USA) on a Bromma 8800 ULTRATOME III ultramicrotome (LKB, Sweden). They were collected on 300 mesh copper grids (Agar Scientific Ltd., Stansted, UK) and double contrasted with saturated alcoholic uranyl acetate (Merck, Darmstadt, Germany) for 12 min, and 2.8% lead citrate (Fluka AG, Buchs, Switzerland). The sections were examined on a JEOL JEM 1010 transmission electron microscope (JEOL Ltd., Japan) at 80 kV, and images were captured using a Mega VIEW III camera (Olympus, Soft Imaging System, Germany).
9
Tissue Preparation for Transmission Electron Microscopy
10
Ultrastructural Analysis of Murine Corpus Callosum
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A subset of C57BL/6J mice at 12 and 12 + 12 week (n = 4–5/timepoint) was transcardially perfused with PBS and fixed with 2% paraformaldehyde and 2% glutaraldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA) for 10 min. The brains were removed and kept in the fixative solution overnight at 4°C. The brains were then segmented at the medial sagittal plane and trimmed into 1–2 mm thickness sagittal sections and kept in 0.1 M sodium cacodylate buffer on ice. After washing, the tissues were post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated in an ascending ethanol series and embedded in either Embed 812 or Araldite epoxy resin (Electron Microscopy Sciences, Hatfield, PA, USA). Tissues were cut into semi-thin sections (400 nm thickness) and stained with Toluidine Blue for visualization, and ROIs were determined. Ultra-thin sections (100 nm thickness) containing the medial mid-caudal corpus callosum were cut with a Leica EM UC6 ultramicrotome and diamond knife (DiATOME; Hatfield, PA, USA), post-stained in 2% aqueous uranyl acetate followed by lead citrate and examined with an FEI Morgagni 268 transmission electron microscope. Digital images were acquired with a MegaViewIII camera operated with iTEM software (Olympus Soft Imaging Systems, Germany).
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