Cochleae were fixed in 2% paraformaldehyde with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 h, washed with 0.1 M phosphate buffer, post-fixed with 1% OsO4 in 0.1 M phosphate buffer for 2 h, dehydrated using ethanol series, and infiltrated with propylene oxide (Fisher scientific, 10,562,351) for 10 min. Specimens were embedded with a Poly/Bed 812 kit (Polysciences, 08792–1) and polymerized in an electron microscope oven (TD-700, DOSAKA, Japan) at 65°C for 12 h. The tissue block was cut into 200-nm semi-thin sections using an ultramicrotome equipped with a diamond knife and stained with toluidine blue for optical microscope observation. The region of interest was then cut into 80-nm sections using the ultramicrotome, placed on copper grids (EMS, EMS300-Cu), double stained with 3% uranyl acetate for 30 min and 3% lead citrate for 7 min, and imaged using a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) equipped with a Megaview III CCD camera (Soft imaging system-Germany) at an acceleration voltage of 80 kV.
Megaview 3 ccd camera
Manufactured by Olympus
Sourced in Germany, Japan, United States
The Megaview III CCD camera is a high-performance imaging device designed for scientific and industrial applications. It features a charge-coupled device (CCD) sensor that captures detailed digital images with high resolution and sensitivity. The camera is capable of capturing images with a wide range of exposure times, making it suitable for a variety of imaging tasks.
Automatically generated - may contain errors
Lab products found in correlation
Jem 1011, by JEOL (11 mentions)
Poly bed 812 kit, by Polysciences (10 mentions)
Cm100 electron microscope, by Thermo Fisher Scientific (4 mentions)
Item software, by Olympus (4 mentions)
Ultracut ultramicrotome, by Leica (3 mentions)
Morgagni 268d electron microscope, by Thermo Fisher Scientific (3 mentions)
Glutaraldehyde, by Agar Scientific (3 mentions)
Analysis camera control software, by Olympus (3 mentions)
Embed 812 araldite, by Electron Microscopy Sciences (2 mentions)
Uranyl acetate, by Agar Scientific (2 mentions)
Epon durkupan, by Merck Group (2 mentions)
Morgagni 268, by Thermo Fisher Scientific (2 mentions)
S 4800 sem, by Hitachi (2 mentions)
Tecnai 12 transmission electron microscope, by Thermo Fisher Scientific (2 mentions)
300 mesh carbon coated copper grid, by Ted Pella (2 mentions)
Diamond knife, by Electron Microscopy Sciences (1 mentions)
Reichert ultracut e, by Leica (1 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (1 mentions)
902 electron microscope, by Zeiss (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
50 protocols using megaview 3 ccd camera
1
Electron Microscopy of Cochlear Tissues
2
Mitochondrial Outer Membrane Integrity Evaluation
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Mitochondrial outer membrane integrity was tested by addition of 10 μM cytochrome c. Since an intact outer mitochondrial membrane provides a complete barrier against penetration of cytochrome c, an increase in respiration after addition of exogenous cytochrome c reflects disruption of outer mitochondrial membrane caused by homogenization and the isolation procedure[25 (link)]. Up to a 10–20% increase in respiration is considered acceptable as evidence of preserved integrity of mitochondrial membrane[26 (link),27 (link)]. Damage to outer mitochondrial membrane was defined as percent of increase in oxygen consumption after addition of exogenous cytochrome c, when both ADP and mitochondrial substrates were present, and calculated as 100*(cyt c-ADP)/ADP. The respiratory control ratio (State 3/State 4), indicating mitochondrial coupling of respiration to phosphorylation, was used as an additional parameter testing functional integrity of mitochondria[26 (link),28 (link)]. The morphology of mitochondria in the homogenate was assessed by electron microscopy using FEI Morgagni 268 transmission electron microscope (see Fig 3 ). The images were captured by Mega View III CCD camera (Olympus Soft Imaging Solutions). More details about preparation of mitochondria for electron microscopy and imaging are described in Supplementary Appendix .
3
Transmission Electron Microscopy Sample Preparation
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Specimens were fixed for 12 h in 2% glutaraldehyde/2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and washed in 0.1 M phosphate buffer, post-fixed with 1% OsO4 in 0.1 M phosphate buffer for 2 h, dehydrated with an ascending ethanol series (50%, 60%, 70%, 80%, 90%, 95%, 100%, and 100%) for 10 min each, and infiltrated with propylene oxide for 10 min.
The fixed samples were embedded using a Poly/Bed 812 kit (Polysciences, Warrington, PA, USA) and polymerized in an electron microscope oven (DOSAKA, Katsumi, Japan) at 65 °C for 12 h. The block was equipped with a diamond knife in the ultramicrotome, cut into 200 nm sections, and stained with toluidine blue for optical microscopy.
The region of interest was then cut into 80 nm sections using the ultramicrotome, placed on copper grids, double stained with 3% uranyl acetate for 30 min and 3% lead citrate for 7 min, and observed under a TEM (JEOL, Tokyo, Japan) equipped with a Megaview III CCD camera (Soft Imaging System-Germany) at an acceleration voltage of 80 kV.
The fixed samples were embedded using a Poly/Bed 812 kit (Polysciences, Warrington, PA, USA) and polymerized in an electron microscope oven (DOSAKA, Katsumi, Japan) at 65 °C for 12 h. The block was equipped with a diamond knife in the ultramicrotome, cut into 200 nm sections, and stained with toluidine blue for optical microscopy.
The region of interest was then cut into 80 nm sections using the ultramicrotome, placed on copper grids, double stained with 3% uranyl acetate for 30 min and 3% lead citrate for 7 min, and observed under a TEM (JEOL, Tokyo, Japan) equipped with a Megaview III CCD camera (Soft Imaging System-Germany) at an acceleration voltage of 80 kV.
4
Ultrastructural Analysis of Newborn Mouse Lungs
5
Zebrafish Larval Ultrastructural Analysis
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Zebrafish larvae were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.02% picric acid, 0.1 M Na cacodylate buffer, washed and fixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer. They were subsequently treated with 0.5% tannic acid followed by 1% sodium sulfate. The pellets were treated with propylene oxide and embedded in Epon/Araldite. Thin sections (70 nm) of the pelleted samples were cut on a Reichert Ultracut E (Leica, Deerfield, IL) using a diamond knife (Diatome, Electron Microscopy Sciences, Hatfield, PA), mounted on parlodion-coated copper slot grids and stained in uranyl acetate and lead citrate. Sections were examined on a Philips CM100 transmission electron microscope (FEI, Hillsbrough, OR). Images were documented and measurements were taken using a Megaview III CCD camera (Olympus Soft Imaging Solutions, Lakewood CO). Transverse sections were obtained through the trunk muscle region, the yolk and the eye region.
6
TEM Imaging of Sample Preparation
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For sample preparation, a drop of sample was placed on the Formvar‐carbon coated grid for 15 s, the droplet was removed using filter paper, a drop of 1% uranyl acetate was put for 15 s, and removed using filter paper, and washed with a drop of distilled water. Dried grids were imaged with transmission electron microscopy (JEM‐1011, JEOL, Tokyo, Japan) at the acceleration voltage of 80 kv equipped with a Megaview III CCD camera (Softimaging system‐Germany).
7
Ultrastructural Analysis of Cells and Tumor Tissues
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For sample preparation, the cells and tumor tissues were fixed in 2.5% glutaraldehyde in PBS (pH 7.4). The samples were further fixed with 1% osmium tetroxide for 1 hour, gradually dehydrated with EtOH gradients, and embedded in epoxy resin. For TEM analysis, sections (70 nm) were cut using a Leica Ultra‐CUT ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) and contrasted with 0.1% lead citrate and 8% uranyl acetate in 50% EtOH. Ultrathin sections were examined using a transmission electron microscope (Tecnai™ G2 Spirit, FEI Company, Hillsboro, OR, USA) that operated at 120 kV. Images were captured using a Megaview III CCD camera (Soft Imaging System, Lakewood, CO, USA).
8
Ultrastructural Analysis of Pancreatic Tissue
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TEM was performed using less than 0.5 mm3 bits of TLCS treated pancreatic fragments as described earlier36 (link). After completion of treatment the pancreatic fragments were immersed in modified Karnovsky’s solution [2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2]. After 48 hrs of Karnovsky fixation tissues were transferred to PBS and transported for transmission electron microscopy. The tissues were post-fixed in 1% OsO4, dehydrated in ascending grades of acetone, embedded and blocked in araldite CY212. After determining the regions of interest on toluidine blue stained sections, 50–60 nm sections were cut on a Reichert-Jung [Leica, Massachussetts, USA] ultracut microtome and collected on 300 mesh copper grids. The sections were stained with uranyl acetate and lead citrate and viewed under Philips Morgagni 268 D TEM [Field Emission Inc., Netherlands]. A MegaView III CCD camera that was integrated with the iTEM software [Olympus Soft Imaging Solutions, Münster, Germany] acquired the photographs, with instrument-calibrated scale bars.
9
Transmission Electron Microscopy Sample Preparation
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Cells were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 h at room temperature. They were then post-fixed for 1 h in 1% osmium tetroxide mixed with 1.5% potassium ferrocyanide in the same buffer. The samples were then exposed to successive baths of increasing ethanol concentration for dehydration and embedded in epoxy resin (Embed 812, EMS 14120). Once the resin had cured, ultra-thin sections (70 nm) were performed and collected on copper grids coated with collodion-carbon (EMS, G200-Cu). They were stained with a 2% aqueous solution of uranyl acetate, followed by a staining with lead citrate in Reynold’s solution. Finally, the sections were observed with the Zeiss 902 electron microscope (Carl Zeiss microscopy GmbH, Jena, Germany), using the Megaview III CCD camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
10
TEM Analysis of Chrysotile-Induced Autophagy in A549 Cells
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