TEM Images were obtained using a FEI Technai G2 Spirit transmission electron microscope (FEI, USA), Macrofire (Optronics) digital camera and AMT image capture Software with assistance from the Campus Microscopy and Imaging Facility (CMIF) at The Ohio State University. Cells were cultured on Permanox (Lab-Tek) chamber slides and fixed with 2.5% gluteraldehyde in 0.1M phosphate buffer with 0.1M sucrose. Slides were post fixed with 1% osmium tetroxide in phosphate buffer then en bloc stained with 2% uranyl acetate in 10% ethanol, dehydrated in a graded series of ethanols and embedded in Eponate 12 epoxy resin (Ted Pella Inc., USA). Ultrathin sections were cut on a Leica EM UC6 ultra microtome (Leica microsystems, Germany), collected on copper grids, and then stained with lead citrate and uranyl acetate.
Technai g2 spirit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Technai G2 Spirit is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of materials at the nanoscale. It features a high-resolution electron optical system and advanced imaging capabilities to enable detailed examination of a wide range of sample types.
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5 protocols using technai g2 spirit
1
Transmission Electron Microscopy of Cells
2
Transmission Electron Microscopy of Infected Macrophages
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TEM Images were obtained using a FEI Technai G2 Spirit transmission electron microscope (FEI, USA), Macrofire (Optronics) digital camera and AMT image capture Software with assistance from the Campus Microscopy and Imaging Facility (CMIF) at The Ohio State University. MDMs were isolated and infected with k56-2 at an MOI of 10 for 1 hour prior to 24 hour experimental treatments. Cells were cultured on Permanox (Lab-Tek) chamber slides and fixed with 2.5% gluteraldehyde in 0.1 M phosphate buffer with 0.1 M sucrose. Slides were post fixed with 1% osmium tetroxide in phosphate buffer then en bloc stained with 2% uranyl acetate in 10% ethanol, dehydrated in a graded series of ethanols and embedded in Eponate 12 epoxy resin (Ted Pella Inc., USA). Ultrathin sections were cut on a Leica EM UC6 ultra microtome (Leica microsystems, Germany), collected on copper grids, and then stained with lead citrate and uranyl acetate.
3
Nanoparticle Imaging via Transmission Electron Microscopy
4
Negative Staining for Electron Microscopy of Extracellular Vesicles
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Samples were fixed with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). EVs were adsorbed onto formvar‐carbon coated grids (EMS) by glow discharging (Pelco EasiGlow) grids and then incubating 7.5 ul of sample on the grids for 20 min at room temperature. Samples were fixed with 1% glutaraldehyde, stained with 1% uranyl acetate and imaged with an FEI Technai G2 Spirit transmission electron microscope (FEI), Macrofire (Optronics) digital camera, and AMT image capture software.
5
Ultrastructural Analysis of Nanoceria
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Animals were fixed, perfused with 2% paraformaldehyde, and intact brains were collected. Hippocampus was microdissected and sectioned into 1×1 mm pieces and allowed to fix overnight in 2.5% gluteraldehyde. Further, tissues were embedded in aryldite resin, cut into 2 nm thin sections using ultramicrotome (Ultramicrotome EM UC7; Leica Microsystems, USA) and fixed on copper grids (Wetzlar, Germany). Grids were analyzed under TEM (Technai G2 Spirit; FEI, OR, USA) at a power of 120 keV and 15,000 magnification. Images were acquired and analyzed using TechnaiEPU automation software (Hillsboro, OR, USA). Presence of nanoceria was further confirmed with SAED pattern. A minimum of two grids per sample were analyzed with five different fields.
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