The central fraction from the GraFix experiment was applied to carbon-coated copper grids and stained with ∼0.75% (w/v) uranyl formate. EM data were collected on a G2 Spirit TEM (FEI Tecnai T12) operated at 120 kV. A total of ∼200 micrograph images were recorded at 50,000× nominal magnification using a CCD camera (Gatan). The micrographs were processed using Relion 3.0.8 software, including particle picking, 2D classification, ab initio reconstruction, and refinement (35 (link)). After few rounds of 2D classification to remove false positive particles, a total of 10,766 particles were used to build the initial model with a C1 symmetry. After 3D classification, a subset of ∼7000 particle in the initial model exhibited nearly a C2 symmetry and were further refined with the C2 symmetry to construct the final 3D EM map at 27 Å.
G2 spirit tem
Manufactured by Thermo Fisher Scientific
The G2 Spirit TEM is a transmission electron microscope (TEM) designed and manufactured by Thermo Fisher Scientific. It is a high-performance, versatile instrument capable of providing detailed images and analysis of materials at the nanoscale level. The core function of the G2 Spirit TEM is to enable users to observe and analyze the internal structure and composition of various samples through the use of an electron beam.
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6 protocols using g2 spirit tem
1
Cryo-EM Structure Determination of Protein Complex
2
Transmission Electron Microscopy of EVs
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EV isolates (10 µg) were added on top of a formvar carbon coated cobber grid for 1 hr. The excess fluid was removed by blotting with a filter paper. The grids were rinsed by dipping in PBS 3 times and dried by a filter paper. The grid with EVs were further fixed by adding a drop of 2.5% glutaraldehyde before washing the grids 5 times with distilled water, and contrasted by adding a drop 2% uranyl acetate. Finally, the grids were rinsed quickly with ice-cold 1.8% methyl cellulose and 0.4% uranyl acetate (MC/UA). The grids were air-dried for 20 min and examined with a FEI Tecnai™ 120 kV transmission electron microscope G2 Spirit TEM (FEI, The Netherlands) equipped with a Morada digital camera and RADIUS imagining software.
3
Cyclo-dipeptide Visualization via TEM
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A carbon-coated copper grid was placed on a 10 µL cyclo-dipeptide droplet for 1 min and then blotted. Next, the washed grid was placed on a 10 µL droplet containing 4% (w/v) uranyl acetate solution for 1 min and then blotted. Samples were examined using Tecnai G2 Spirit TEM (FEI) at 80 kV.
4
Organoid Ultrastructural Characterization
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Therminox coverslips (Ted Pella 26028) were sterilized with 70% ethanol and UV irradiation for 1 h prior to coating in the usual fashion (Matrigel for Y6 iPSCs; vitronectin for ACS1028 iPSCs; and fibronectin for iPSC-ECs). Cells were plated and grown as described. At the desired time intervals, the coverslips were removed and placed in warmed (0.1M sodium cacodylate, pH 7.4, 2% paraformaldehyde, and 2.5% glutaraldehyde) and fixed for 60 min at 37°. Cells were then post-fixed in 2% osmium tetraoxide in imidazole buffer 0.1M (pH 7.5) for 1 h, rinsed in distilled water, stained with 3% UA for 1 h, and again rinsed in distilled water. Samples were then dehydrated in ascending grades of acetone, acetone and resin, embedded in a mixture of an Embed 812 kit (EMS 14120), and cured for 48 h in a 60 °C oven.
Ultrathin sections (70 nm) of the organoid were produced using a Leica UltraCut UC6 ultramicrotome, collected on 200 mesh copper grids and post-stained with 3% UA and Reynolds lead citrate. Micrographs were collected using on an FEI Technai Spirit G2 TEM.
Ultrathin sections (70 nm) of the organoid were produced using a Leica UltraCut UC6 ultramicrotome, collected on 200 mesh copper grids and post-stained with 3% UA and Reynolds lead citrate. Micrographs were collected using on an FEI Technai Spirit G2 TEM.
5
TEM and NMR Analysis of PA-Losartan Complex
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Samples for transmission electron microscopy (TEM) were resuspended at a PA concentration of 10 mg/mL and then diluted 10-fold–1 mg/mL immediately before 10 µL of sample solution was transferred to plasma-cleaned 300-mesh copper grids with lacey carbon support (Electron Microscopy Science). Samples were stained with 2% uranyl acetate. Imaging was performed using a FEI Spirit G2 TEM working at 120 kV accelerating voltage.
Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Neo 600 MHz system with QCI-F cryoprobe at 298 K. The lyophilized powders from the sample preparation protocol above were resuspended in D2O at 10 mg/mL PA concentration and 5 mg/mL losartan concentration. This corresponds to 8.7 mM E-PA, 8.7 mM K-PA, and 10.8 mM losartan.
Nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker Neo 600 MHz system with QCI-F cryoprobe at 298 K. The lyophilized powders from the sample preparation protocol above were resuspended in D2O at 10 mg/mL PA concentration and 5 mg/mL losartan concentration. This corresponds to 8.7 mM E-PA, 8.7 mM K-PA, and 10.8 mM losartan.
6
Characterization of PA Nanofibers by TEM
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