Ultrathin (50–60nm) sections were obtained using a diamond knife (Diatome) and Reichert Ultracut E microtome. Sections were collected on coated copper grids and post-stained with 2.5% uranyl acetate for 10 min. Sections were imaged at 120 kV using a FEI Tecnai 12 transmission electron microscope.
Tecnai 12 transmission
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The Tecnai 12 is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of samples at the nanoscale. It is capable of providing detailed structural and compositional information about a wide range of materials and biological specimens.
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3 protocols using tecnai 12 transmission
1
Ultrathin Sectioning and Imaging
2
Transmission Electron Microscopy Specimen Preparation
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For negative staining, cell culture supernatants were absorbed on Formvar and carbon-coated grids for 1 min and stained with 1% (w/v) phosphotungstic acid (pH 6.8) for 1 min. Grids were air dried and observed under a Tecnai 12 transmission electron microscope (FEI, Eindhoven, Netherlands).
For ultrathin sections, cell pellets were fixed in a solution of 2% formaldehyde and 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in an ethanol gradient, embedded in epoxy resin, and polymerized at 60 °C for 24 h. Ultrathin sections (80 nm) were obtained from the resin blocks, mounted on copper grids, and stained with uranyl acetate and lead citrate. Finally, the ultrathin sections were observed using transmission electron microscopy.
For ultrathin sections, cell pellets were fixed in a solution of 2% formaldehyde and 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in an ethanol gradient, embedded in epoxy resin, and polymerized at 60 °C for 24 h. Ultrathin sections (80 nm) were obtained from the resin blocks, mounted on copper grids, and stained with uranyl acetate and lead citrate. Finally, the ultrathin sections were observed using transmission electron microscopy.
3
Transmission Electron Microscopy Sample Preparation
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The resin-embedded cells were cut into ultrathin sections using a diamond knife. Sectioned samples were inserted into a Pelco grid staining system. Samples were stained in 2% methanolic uranyl acetate (2% uranyl acetate dissolved in 70% methanol–H2O) for 5 min at room temperature, washed 5 times in DI water, and then stained in Reynolds lead citrate (recipe for 50 ml: 1.33 g lead nitrate and 1.76 g of sodium citrate dihydrate were dissolved in 30 ml DI H2O; 8 ml of 1 N NaOH was added, followed by 10 ml of DI H2O) for 5 min at room temperature. A total of 10 more washes were performed with DI water, and the sample was dried with a Kimwipe and imaged on an FEI Tecnai 12 transmission electron microscope. The images were cropped, and the size of each image was compressed using Adobe Photoshop. ImageJ was used to add the scale bar.
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