TEM imaging was performed according to the procedure described by Adda et al. (2009) (link). In brief, samples (10 μl) were applied to 400-mesh copper grids coated with a thin layer of carbon for 2 min. Excess material was removed by blotting and samples were negatively stained twice with 10 μl of a 2% (wt/vol) uranyl acetate solution (Electron Microscopy Services, Hatfield, PA). The grids were air-dried and viewed using a JEOL JEM-2010 transmission electron microscope operated at 80 kV.
Uranyl acetate solution
Manufactured by Electron Microscopy Sciences
Sourced in United States
2% uranyl acetate solution is a staining solution commonly used in electron microscopy. It is a heavy metal salt that provides contrast enhancement by binding to biological macromolecules, enabling better visualization of cellular structures during electron microscope imaging.
Automatically generated - may contain errors
Lab products found in correlation
Veleta, by Olympus (4 mentions)
Jem 2010, by JEOL (3 mentions)
Jem 2010 transmission electron microscope, by JEOL (2 mentions)
2100 transmission electron microscope, by JEOL (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Tecnai spirit, by Thermo Fisher Scientific (2 mentions)
Morada, by Olympus (2 mentions)
Dimethylformamide, by Thermo Fisher Scientific (1 mentions)
Dichloromethane, by Thermo Fisher Scientific (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Diethyl ether, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
P toluenesulfonic acid, by Thermo Fisher Scientific (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
2 2 dimethoxypropane, by Thermo Fisher Scientific (1 mentions)
N methylmorpholine, by Thermo Fisher Scientific (1 mentions)
Triisopropylsilane tips, by Thermo Fisher Scientific (1 mentions)
Tecnai tf30, by Thermo Fisher Scientific (1 mentions)
Sodium phosphate dibasic na2hpo4, by Merck Group (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
21 protocols using uranyl acetate solution
1
TEM Imaging of Biological Samples
2
Transmission Electron Microscopy of MEVs
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JEM-2010 transmission electron microscope (, 80 kV, JEOL, Tokyo, Japan) was employed to examine 0.2 mg/mL MEVs. The samples were fixed in mesh carbon-layered copper grids (400) for 2 min and the surplus preparations were drained using blotting paper. Samples were then stained with 10 µL of 2% (w/v) uranyl acetate solution (Electron Microscopy Services) before imaging.
3
Peptide Synthesis and Characterization
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The following reagents were purchased from commercial vendors (Fisher Scientific (Waltham, MA, USA) or Sigma Aldrich (St. Louis, MO, USA)) and used without further purification: guanosine (Fisher Scientific), p-toluenesulfonic acid (Fisher Scientific), 2,2-dimethoxypropane (Fisher Scientific), acetone, (Fisher Scientific), 2,2,6,6,-tetramethyl-1-piperidinyloxyl (Fisher Scientific), [bis(acetoxy-iodo]benzene (Fisher Scientific), dimethylformamide (Fisher Scientific), Fmoc-Phe wang resin (Fisher Scientific), N,N,N′,N′-tetremethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate (HBTU) (TCI America, Tokyo, Japan), fluorenylmethyloxycarbonyl (Fmoc) protected amino acids (Chem-Impex Int., Inc., Wood Dale, IL, USA), N-methylmorpholine (Fisher Scientific), dichloromethane (Fisher Scientific), N,N′-diisopropylcarbodiimide (DIC) (Fisher Scientific), ethyl cyano(hydroxylmino)acetate (Oxyma Pure) (Sigma Aldrich), trifluoroacetic acid (TFA) (Fisher Scientific), triisopropylsilane (TIPS) (Fisher Scientific), diethyl ether (Fisher Scientific), acetonitrile (Fisher Scientific), alpha-cyano-4-hydroxycinnamic acid (CHCA) (Ricca Chemical, Arlington, TX, USA), 4% uranyl acetate solution (Electron Microscopy Sciences). All water used to prepare assembly solutions was 18.2 MΩ ultrapurified water.
4
Transmission Electron Microscopy of EVs
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Microscopy was performed as described previously46 . EV samples (0.2 μg/μL each) were examined with a JEM-2010 transmission electron microscope (JEOL, 100 kV) or Tecnai TF30 transmission electron microscope (FEI, 300 kV). Preparations were fixed to 400 mesh carbon-layered copper grids for up to 2 min. Surplus material was drained by blotting, followed by negative staining of samples with 10 μL of uranyl acetate solution (2% w/v; Electron Microscopy Services).
5
Fmoc-protected Peptide Synthesis
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Fmoc-protected amino acids for peptide synthesis
were purchased from AGTC Bioproducts Ltd. Wang resins were used for
peptide synthesis. Sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), and Thioflavin T were purchased from Sigma-Aldrich (UK). Carbon
grid (200 mesh copper) and uranyl acetate solution were purchased
from Electron Microscopy Sciences. Milli-Q water (18.2 MΩ.cm)
was used for all the experiments.
were purchased from AGTC Bioproducts Ltd. Wang resins were used for
peptide synthesis. Sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), and Thioflavin T were purchased from Sigma-Aldrich (UK). Carbon
grid (200 mesh copper) and uranyl acetate solution were purchased
from Electron Microscopy Sciences. Milli-Q water (18.2 MΩ.cm)
was used for all the experiments.
6
TEM Specimen Preparation and Imaging
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For each TEM sample, a 10-μL droplet of suspension or solution was applied to a carbon-coated Formvar nickel grid (200 mesh) (Electron Microscopy Sciences, Washington, PA, USA). Each sample was allowed to sediment onto the carbon film for 15 min, then negative staining was performed with 10 μL of 2% w/v uranyl acetate solution (Electron Microscopy Sciences). After carefully draining off excess staining solution using filter paper, the specimen was transferred to a Philips CM12 TEM for examination, operating the TEM at 80 kV. Electron micrographs of negatively stained samples were photographed using Kodak film.
7
Transmission Electron Microscopy of Samples
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Samples (0.2 μg/μL) were examined in a JEM-2010 transmission electron microscope (JEOL, 80 kV) or Tecnai TF30 transmission electron microscope (FEI, 300 kV). Preparations were fixed to 400 mesh carbon-layered copper grids for up to 2 min. Surplus material was drained by blotting followed by negatively staining of samples with 10 μL of uranyl acetate solution (2% w/v; Electron Microscopy Services).
8
Characterization of DIV3(X)-siRNA Complexes
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DIV3(X)-siNT complexes were prepared at 2 mg/mL in RNase-free water as previously described. Hydrodynamic size and surface charge of the peptide/siRNA complexes were determined using a Nano ZS Zetasizer (Malvern Pananalytical, Malvern, UK). All data were recorded using Malvern’s Zetasizer software.
In order to obtain TEM images of the peptide-siRNA complexes, DIV3W-siNT complexes were prepared as previously described to a concentration of 1 mg/mL in RNase-free water. After a 20-min incubation, TEM samples were prepared via drop casting onto a copper mesh grid. Following a 5-min drying period, the remaining water was wicked away, and the sample was background stained with 1% uranyl acetate solution (Electron Microscopy Sciences, Hatfield, PA). Samples were stored for 48 h to allow for complete solution evaporation and sample crystallization. TEM was completed using a Hitachi HT7800 microscope (Hitachi, White Plains, NY) and imaged at 80,000× magnification.
In order to obtain TEM images of the peptide-siRNA complexes, DIV3W-siNT complexes were prepared as previously described to a concentration of 1 mg/mL in RNase-free water. After a 20-min incubation, TEM samples were prepared via drop casting onto a copper mesh grid. Following a 5-min drying period, the remaining water was wicked away, and the sample was background stained with 1% uranyl acetate solution (Electron Microscopy Sciences, Hatfield, PA). Samples were stored for 48 h to allow for complete solution evaporation and sample crystallization. TEM was completed using a Hitachi HT7800 microscope (Hitachi, White Plains, NY) and imaged at 80,000× magnification.
9
Visualization of LL-37 Membrane Interactions
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Neat LL-37 as well as membrane mixtures with 10 μM LL-37 (10 μL) were applied for 2 min to 400-mesh copper grids (ProSciTech) coated with a thin layer of carbon. Excess material was removed by blotting, and samples were negatively stained twice with 10 μL of a 2% (wt/vol) uranyl acetate solution (Electron Microscopy Sciences, Hatfield PA, USA). The grids were air-dried and viewed using a JEOL JEM-2010 transmission electron microscope operated at 80 kV.
10
Characterization of Lipid-Polymer Nanocarriers
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The hydrodynamic size, polydispersity index (PDI), and Z-potential were studied for the LPNCs through Dynamic Light Scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). A dilution of 1:10 in milliQ water was performed to adjust the intensity (attenuator position between 3 and 6).
The LPNCs’ morphology, size, and distribution were studied using Transmission Electron Microscopy (TEM). The LPNCs’ size was also measured using the particle analysis tool of the Image J software. The TEM grids were coated with formvar of a 1:10 dilution of LPNCs in milliQ water and incubated for 1 min at room temperature. The grids were washed with milliQ water and stained with a 2% w/w uranyl acetate solution (Electron Microscopy Sciences, Hatfield, England) during 1 min at room temperature. They were then dried in a petri dish overnight and observed using a TEM microscope (Jeol JEM 1010 100 kv; Jeol, Tokyo, Japan).
The LPNCs’ morphology, size, and distribution were studied using Transmission Electron Microscopy (TEM). The LPNCs’ size was also measured using the particle analysis tool of the Image J software. The TEM grids were coated with formvar of a 1:10 dilution of LPNCs in milliQ water and incubated for 1 min at room temperature. The grids were washed with milliQ water and stained with a 2% w/w uranyl acetate solution (Electron Microscopy Sciences, Hatfield, England) during 1 min at room temperature. They were then dried in a petri dish overnight and observed using a TEM microscope (Jeol JEM 1010 100 kv; Jeol, Tokyo, Japan).
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