A suspension of Rotavirus DLPs (strain SA11) was prepared using a previously described method17 (link). The DLPs/AuNPs suspension (4 μl) was placed onto holey carbon EM grids (Quantifoil). Each grid was blotted for 5 s and subsequently plunged into a liquid ethane/propane mixture cooled by liquid nitrogen at 80% humidity. The grids were then transferred and stored under liquid nitrogen. Further details of the sample preparation are provided in Supplementary Text 1 .
Holey carbon em grids
Manufactured by Quantifoil
Sourced in Germany
Holey carbon EM grids are a type of sample support used in electron microscopy (EM) applications. They consist of a thin carbon film with regularly spaced perforations or 'holes' that allow the electron beam to pass through the sample, enabling high-resolution imaging.
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Lab products found in correlation
3 protocols using holey carbon em grids
1
Cryo-EM Sample Preparation of Rotavirus DLPs
2
Cryo-ET of Mitochondrial Ultrastructure
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Holey carbon EM grids (Quantifoil, Jena, Germany) were glow-discharged under vacuum using an ELMO TM Glow Discharge System (Cordouan Technologies, France) according to the manufacturer's instructions. The isolated mitochondrial suspension (5–10 mg/ml) was mixed 1:1 with a stock solution of 10 nm protein A–gold fiducial markers (Aurion, Wageningen, The Netherlands), and 3 µl was immediately applied to a glow-discharged grid. Grids were blotted for 1–1.5 s, followed by plunge-freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Samples were placed in a grid storage box and stored under liquid nitrogen prior to viewing.
Cryo-ET was performed using a FEITM TalosTM, equipped with a 200 kV FEG (Thermo Fisher Scientific), K2 DED and GIF BioQuantum LS energy filter operated with a slit width of 20 eV (Gatan, Pleasanton, USA). Dose-fractionated tomograms (2–5 frames per tilt) were collected at a magnification of 49k× (corresponding to a pixel size of 2.8 Å), typically collected from +60° to −60° with tilt steps of two degrees. The total dose per tomogram was <120 e−/Å2. Gold fiducial markers were used to align tomograms, and volumes were reconstructed using the IMOD software (Kremer et al., 1996 (link)). Tomogram contrast was enhanced by non-linear anisotropic diffusion (NAD) filtering in IMOD.
Cryo-ET was performed using a FEITM TalosTM, equipped with a 200 kV FEG (Thermo Fisher Scientific), K2 DED and GIF BioQuantum LS energy filter operated with a slit width of 20 eV (Gatan, Pleasanton, USA). Dose-fractionated tomograms (2–5 frames per tilt) were collected at a magnification of 49k× (corresponding to a pixel size of 2.8 Å), typically collected from +60° to −60° with tilt steps of two degrees. The total dose per tomogram was <120 e−/Å2. Gold fiducial markers were used to align tomograms, and volumes were reconstructed using the IMOD software (Kremer et al., 1996 (link)). Tomogram contrast was enhanced by non-linear anisotropic diffusion (NAD) filtering in IMOD.
3
Cryo-ET of Mitochondrial Ultrastructure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Holey carbon EM grids (Quantifoil, Jena, DE) were glow-discharged under vacuum using an ELMO TM Glow Discharge System (Cordouan Technologies, FR) according to manufacturer's instructions. The isolated mitochondrial suspension (5-10 mg/mL) was mixed 1:1 with a stock solution of 10 nm protein A-gold fiducial markers (Aurion, Wageningen, NL) and 3 l immediately applied to a glow-discharged grid. Grids were blotted for 1-1.5 sec, followed by plunge-freezing in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Samples were placed in a grid storage box and stored under liquid nitrogen prior to viewing.
Cryo-ET was performed using a FEITM TalosTM, equipped with a 200 kV FEG (Thermo Fisher Scientific), K2 DED and GIF BioQuantum LS energy filter (Gatan, Pleasanton, USA). Dosefractionated tomograms were typically collected from +60°to 60° with tilt steps of two degrees. Gold fiducial markers were used to align tomograms and volumes were reconstructed using the IMOD software [71] . Tomogram contrast was enhanced by non-linear anisotropic diffusion (NAD) filtering in IMOD.
Cryo-ET was performed using a FEITM TalosTM, equipped with a 200 kV FEG (Thermo Fisher Scientific), K2 DED and GIF BioQuantum LS energy filter (Gatan, Pleasanton, USA). Dosefractionated tomograms were typically collected from +60°to 60° with tilt steps of two degrees. Gold fiducial markers were used to align tomograms and volumes were reconstructed using the IMOD software [71] . Tomogram contrast was enhanced by non-linear anisotropic diffusion (NAD) filtering in IMOD.
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