Electron microscopy was performed at the EMBION facility (embion.au.dk), iNANO, Aarhus University. 3 μL of a diluted sample was added to a 400 mesh collodion (Sigma Aldrich) and carbon coated copper grid (Pelco) that had been glow discharged 45s at 25 mA and 39 mbar using an EasiGlow (Pelco). After a 30s incubation, the grid was blotted using a 85 mm filter paper grade 1 (lot. no. 10302, Whatman) and washed/stained 3x with 3 µL 2% uranyl formate (Polysciences Europe GmbH) with blotting steps between each washing/staining step and a final blotting and drying step. Micrographs were collected using a Tecnai Spirit TWIN transmission electron microscope (ThermoFisherScientific) operated at 120 kV using a TemCam F416 CMOS camera and EM-Menu software (Tvips).
Tecnai spirit twin transmission electron microscope
Manufactured by Thermo Fisher Scientific
The Tecnai Spirit Twin transmission electron microscope is a high-performance instrument designed for advanced imaging and analysis of materials at the nanoscale level. It features a twin-lens configuration that provides enhanced resolution and flexibility in imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
400 mesh collodion, by Merck Group (2 mentions)
Em menu software, by TVIPS (2 mentions)
Temcam f416 cmos camera, by TVIPS (2 mentions)
Uranyl formate, by Polysciences (2 mentions)
Millonig s sodium phosphate buffer, by Tousimis (2 mentions)
Lead citrate, by Merck Group (1 mentions)
Lr white resin, by Ted Pella (1 mentions)
Uc7 ultramicrotome, by Leica camera (1 mentions)
6 protocols using tecnai spirit twin transmission electron microscope
1
Negative Staining Electron Microscopy
2
Isolation and Characterization of T-EVs
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T‐EVs isolated from app. 25 × 106 activated human CD4+ T cells, were washed in PBS and ultracentrifuged a second time at 100,000 × g. The T‐EVs were diluted in 200 μL PBS and stored at −80°C for 1 month prior to analysis by electron microscopy. Electron microscopy was performed at the EMBION facility (embion.au.dk), iNANO, Aarhus University. Three microliters of a diluted sample was added to a 400 mesh collodion (Sigma Aldrich) and carbon coated copper grid (Pelco) that had been glow discharged 45 s at 25 mA and 39 mbar using an EasiGlow (Pelco). After a 30 s incubation the grid was blotted using a 85 mm filter paper grade 1 (lot. no. 10302, Whatman) and washed/stained 3× with 3 μL 2% uranyl formate (Polysciences Europe GmbH) with blotting steps between each washing/staining step and a final blotting and drying step. Micrographs were collected using a Tecnai Spirit TWIN transmission electron microscope (ThermoFisherScientific) operated at 120 kV using a TemCam F416 CMOS camera and EM‐Menu software (Tvips).
3
Transmission Electron Microscopy of Fly Heads
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Adult fly heads were dissected, fixed, dehydrated, and embedded in LR White resin (Ted Pella, Redding, CA) as previously described (Xu and Wang, 2016 (link)). Thin sections (80 nm) prepared at a depth of 30–40 μm were stained with uranyl acetate and lead citrate (Sigma, St. Louis, MO) and examined using a FEI Tecnai Spirit Twin transmission electron microscope (FEI, Hillsboro, OR).
4
Negative Stain Electron Microscopy
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Electron microscopy measurements were performed by Electron Microscopy BioServices (Frederick, MD) using a FEI Tecnai Spirit Twin Transmission Electron Microscope (FEI, OR) at high magnifications at 80 kV. Negative stain was performed by incubation for 60 s with 2% phosphotungstic acid. Excess stain was removed with filter paper.
5
Electron Microscopy Analysis of EBOV-Infected iPSC-HLCs
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For conventional thin-section microscopic evaluation, 4 × 105 EBOV-infected BU1, BU2, and BU3 iPSC-HLCs were inactivated and preserved in 2.5% glutaraldehyde (E.M. Sciences, Warrington, PA), in Millonig’s sodium phosphate buffer (Tousimis Research, Rockville, MD), for 24 h (n = 1 for each donor at each time point for a total of three samples per time point). After fixation was complete, the cells were washed in Millonig’s buffer, and incubated for 2 h in 1.0% osmium tetroxide, in the same buffer. Following rinsing steps in ultrapure water and en bloc staining with 2.0% uranyl acetate, the samples were dehydrated in a series of graded ethanol washes and infiltrated and embedded in Spurr’s plastic resin (E.M. Sciences). Embedded blocks were sectioned using a Leica UC7 Ultramicrotome, and 70- to 80-nm sections were collected on 200 mesh copper grids, and post-stained with Reynold’s lead citrate. Samples were examined in an FEI Tecnai Spirit Twin transmission electron microscope, operating at 80 kV.
6
Viral Quantitation via Negative Staining
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For negative staining analysis and viral quantitation, cell culture supernatant was collected from infected Vero E6 cells clarified by low speed centrifugation, and inactivated by diluting in an equal volume of 8.0% paraformaldehyde (PFA) (E.M. Sciences, Warrington, PA), in Millonig’s sodium phosphate buffer (Tousimis Research, Rockville, MD) for a final concentration of 4% PFA.
After inactivation, 50 μL of supernatant was mixed with an equal volume of latex beads (Structure Probe Inc., West Chester, PA) of a known concentration, and 2 μL of the supernatant/latex bead samples were mounted on individual 300 mesh copper grids reinforced with formvar resin and carbon, allowed to dry, and stained with 1.0% Phosphotungstic Acid (E.M. Sciences). Specimen grids were examined in a Tecnai Spirit Twin Transmission Electron Microscope, (FEI, Hillsboro, OR) operating at 80 kV.
For virus quantitation, 1000 latex beads were enumerated over eight different grid openings (average of 125 beads per opening), along with intact NiV particles. Using ratio formulas, the concentration of each viral supernatant per mL was then calculated.
After inactivation, 50 μL of supernatant was mixed with an equal volume of latex beads (Structure Probe Inc., West Chester, PA) of a known concentration, and 2 μL of the supernatant/latex bead samples were mounted on individual 300 mesh copper grids reinforced with formvar resin and carbon, allowed to dry, and stained with 1.0% Phosphotungstic Acid (E.M. Sciences). Specimen grids were examined in a Tecnai Spirit Twin Transmission Electron Microscope, (FEI, Hillsboro, OR) operating at 80 kV.
For virus quantitation, 1000 latex beads were enumerated over eight different grid openings (average of 125 beads per opening), along with intact NiV particles. Using ratio formulas, the concentration of each viral supernatant per mL was then calculated.
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