MACs assembled on lipid monolayers were imaged using EM, as previously described (42 (link)). 10 μl of buffer (120 mm NaCl, 20 mm Hepes pH 7.4) were deposited in a 4 mm diameter well teflon plate and overlaid with 2 μl of lipids in chloroform [DOPC:DOPE 60:40 molar ratio, 1 mg/ml (Avanti Polar Lipids, Alabama, USA)]. The chloroform was evaporated for 1 min and an EM grid (CF400-CU, Agar Scientific, Stansted, UK) was placed carbon-side down onto the solution surface. MAC components were sequentially added to the solution with minimal perturbation as follows: C5b6, C7, C8 [60 nm , 5 min incubation between each new addition, 37°C (Complement Technologies, Texas, USA)] and C9 (1.2 μm , 15 min incubation, 37°C) to achieve a final molar ratio of 1:1:1:20. The grid was then gently peeled off and immediately stained with 2% uranyl acetate. Images were acquired on a Tecnai G2 Spirit electron microscope (Thermo Fisher Scientific, Paisley, UK) with a 2K eagle camera (Thermo Fisher Scientific, Paisley, UK) at ×42 000 magnification (3.7 A/px).
Tecnai g2 spirit electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Tecnai G2 Spirit electron microscope is a high-performance transmission electron microscope (TEM) designed for advanced imaging and analysis applications. It utilizes an electron beam to magnify and focus on samples, providing detailed information about their structure and composition at the nanoscale level.
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Lab products found in correlation
Ultracut uct ultramicrotome, by Leica (4 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Quemesa, by Olympus (3 mentions)
Whatman 4 filter paper, by Cytiva (2 mentions)
Glow discharge cleaned copper grid, by Electron Microscopy Sciences (2 mentions)
Morada, by Olympus (2 mentions)
Ab23792, by Abcam (2 mentions)
Z0412, by Agilent Technologies (2 mentions)
Sc 1000 orius ccd camera, by Adobe (2 mentions)
Jem 1400 transmission electron microscope, by JEOL (2 mentions)
Zetaview, by Particle Metrix (1 mentions)
Ultrafiltration tube, by Merck Group (1 mentions)
Ultrascan ccd camera, by Ametek (1 mentions)
Lacey formvar carbon 200 mesh copper grids, by Ted Pella (1 mentions)
P804536, by Maclin Biochemical Technology (1 mentions)
G5882, by Merck Group (1 mentions)
Cryo diamond knife, by Electron Microscopy Sciences (1 mentions)
200 mesh copper em grids, by ProSciTech (1 mentions)
A1 confocal system, by Nikon (1 mentions)
Epon 812 resin, by Electron Microscopy Sciences (1 mentions)
58 protocols using tecnai g2 spirit electron microscope
1
Imaging MAC Assembly on Lipid Monolayers
2
Exosome Isolation and Characterization
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Exosomes were collected from the macrophage culture medium (CM) through ultracentrifugation. To remove cells and debris, the cell culture medium was centrifuged at 300 g for 5 min and 3000 g for 30 min. The medium was then added to ultrafiltration tubes (100 kDa, Merck Millipore, USA) and centrifuged at 4000 g for 5 min. The exosomes were collected by centrifugation at 150,000 g for 2 h. After resuspension in PBS the exosomes were purified again through ultracentrifugation.
The size and shape of exosomes were verified through transmission electron microscopy (TEM). Exosomes were diluted 100 times using PBS, dropped into carbon-coated copper grids and stained with 2% glutaraldehyde. After the copper grids had dried, the Tecnai G2 Spirit electron microscope (Thermo Fisher Scientific, USA) was used to capture stained images.
The sizes and concentrations of exosomes were detected through nanoparticle tracking analysis (NTA) and calculated using ZetaView (Particle Metrix, Germany). Exosomes were diluted 500 times by PBS and fully resuspended before being injected into the sample pool. According to the measured concentrations, the exosome solutions of different samples were adjusted to the same concentration by PBS.
The size and shape of exosomes were verified through transmission electron microscopy (TEM). Exosomes were diluted 100 times using PBS, dropped into carbon-coated copper grids and stained with 2% glutaraldehyde. After the copper grids had dried, the Tecnai G2 Spirit electron microscope (Thermo Fisher Scientific, USA) was used to capture stained images.
The sizes and concentrations of exosomes were detected through nanoparticle tracking analysis (NTA) and calculated using ZetaView (Particle Metrix, Germany). Exosomes were diluted 500 times by PBS and fully resuspended before being injected into the sample pool. According to the measured concentrations, the exosome solutions of different samples were adjusted to the same concentration by PBS.
3
Transmission Electron Microscopy of Oviduct Tissue
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TEM was performed as described by Bathala et al. [5 (link)]. One oviduct tissue representative of each estrous cycle stage was fixed with filtered PBS containing 2% formaldehyde and 2% glutaraldehyde and stored at 4 °C. The tissues were treated with 1% osmium acid for 1 h, washed three times (10 min each) with water, and treated with 2% uranium acetate solution. Thereafter, the tissues were dehydrated using the graded series of ethanol (50%, 70%, 90%, 100% ethanol) and 100% acetone. The tissues were then treated with a mixture of embedding agent and acetone (1:1 at room temperature for 2 h, and 1:3 at room temperature for 3 h). Ultrathin sections of the tissues after polymerization were observed using a Tecnai G2 Spirit electron microscope (Thermo Fisher Scientific) operated at 120 kV.
4
Ultrastructural Analysis of Mouse Cardiac Mitochondria
5
Cryogenic Imaging of Extracellular Vesicles
6
Retina Tissue Ultrastructural Analysis
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Retina samples were collected and fixed in 2.5% glutaraldehyde (G5882, Sigma-Aldrich) and 4% formaldehyde (P804536, Macklin) in 0.1 M HEPES buffer for 4 to 5 h. After washing in 0.1 M phosphate buffer three times, tissues were fixed with 1% osmic acid at 4 °C for 2 h. Tissues were washed in 0.1 M phosphate buffer three times, dehydrated using a graded alcohol series, and embedded in epoxy resin. Samples were examined under a Tecnai G2 Spirit electron microscope (FEI).
7
Cryo-Sectioning and Electron Microscopy
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Blocks were transferred to aluminum cryo-sectioning pins (Leica) and quickly plunge-frozen in liquid nitrogen. Thin cryo-sections (80 nm) were cut at −100°C with an EM-UC6/FC7 cryo-ultramicrotome (Leica) using a cryo-diamond knife (Diatome). Cryo-sections were removed from the knife with 2.3 M sucrose using a wire loop and transferred to formvar/carbon-coated, plasma cleaned, 200-mesh copper EM grids (Proscitech). Grids were stored in an airtight container on sucrose droplets at 4°C. To stain, grids were floated face down on 2% gelatin for 30 min at 37°C before washing in PBS (3 min × 2 min) and staining with 2% uranyloxalicacetate, pH 7 (5 min, room temperature) and methyl cellulose–uranyl acetate pH 4 on ice (10 min). Grids were looped out, drained, and allowed to dry. Samples were imaged with a Tecnai G2 Spirit electron microscope (FEI Company) operated at 100 kV at Adelaide Microscopy, the University of Adelaide, South Australia.
8
Exosomal Morphology Visualization
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The exosomal pellets were resuspended in 50 μL phosphate-buffered saline and a drop of the suspension was placed on a sheet of parafilm. A copper grid was floated on the drop for 2 minutes at room temperature and was subsequently touched onto a drop of 2% phosphotungstic acid and stained for 2 minutes. The grid was allowed to dry for several minutes and was examined. The images were captured using a Tecnai G2 Spirit electron microscope (FEI Co., Hillsboro, OR, USA).
9
Electron Microscopy Protocol for Cell Analysis
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Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, embedded in 2% agarose, postfixed in buffered 1% osmium tetroxide, en bloc stained in 4% uranyl acetate, dehydrated with a graded series of ethanol, and embedded in EMbed-812 resin. Thin sections were cut on a Leica Ultracut UCT ultramicrotome and stained with 2% uranyl acetate and lead citrate. Images were acquired on a FEI Tecnai G2 Spirit electron microscope equipped with a LaB6 source and operating at 120 kV.
10
Confocal Imaging of Fluorescently Labeled Samples
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Digital images of fluorescent labeling were collected using Nikon A1 confocal system (Japan) equipped with Nikon 90i upright fluorescence microscope and BioRad Laser Sharp 2000 imaging program (Digital BioRad Center, Pleasanton, CA, USA). Alexa Fluor 568 labeling was viewed through 561 excitation laser line with 10 nm resolution of spectra. Alexa Fluor 488 and ByLight 405 were viewed through 488 and 405 excitation laser line, also through 10 nm resolution spectra. Confocal images were captured with ×20 and ×40 objective at iris of 2.0–2.5 in box size of 1,024×1,024. All Z-scan was set up at 1 μm layer of laser scan step and saved as “avi” video files and the clearest double or triple labeling image was selected and converted to Photoshop 7.0.1 (Adobe, CA, USA) through Bio-Rad Plug-In software and stored as “tiff” file at 1,024×1,024 pixels. The electron microscopy microphotographs presented here were taken with FEI Tecnai G2 Spirit electron microscope.
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