Gatan 626 cryo holder

Manufactured by Thermo Fisher Scientific

The Gatan 626 cryo-holder is a specialized lab equipment designed for cryogenic electron microscopy (cryo-EM) applications. It is used to hold and stabilize samples at cryogenic temperatures, enabling the examination of biological specimens in their native state.

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Lab products found in correlation

Tecnai t20, by Thermo Fisher Scientific (1 mentions) Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions) Eagle 4k 4k camera, by Thermo Fisher Scientific (1 mentions) Leica gp plunger, by Leica camera (1 mentions) Talos electron microscope, by Thermo Fisher Scientific (1 mentions) Tecnai 12 electron microscope, by Thermo Fisher Scientific (1 mentions) Em gp, by Leica camera (1 mentions) Falcon 3 camera, by Thermo Fisher Scientific (1 mentions) Em pc cryo system, by Leica camera (1 mentions) Tecnai f20 cryo electron microscope, by Thermo Fisher Scientific (1 mentions)

5 protocols using gatan 626 cryo holder

Cryo-TEM Peptide Capsid Visualization

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Cryo-TEM samples were prepared by plunge freezing performed using a VITROBOT mark IV (FEI Company)—into liquid N₂-cooled liquid ethane. Droplets (5 µL) of peptide solutions (100 µM) were placed on glow discharged lacey carbon grids and left for 2 s before blotting (2 s) and plunging. This yielded samples in which peptide capsids are embedded in the vitreous ice suspended inside the holes of the carbon. The sample grid was then transferred (without warming) into a Gatan 626 cryo-holder and visualised at 200 kV in a Tecnai T20 (FEI company) transmission electron microscope fitted with an Eagle 4k × 4k camera (FEI).

De Santis E., Alkassem H., Lamarre B., Faruqui N., Bella A., Noble J.E., Micale N., Ray S., Burns J.R., Yon A.R., Hoogenboom B.W, & Ryadnov M.G. (2017). Antimicrobial peptide capsids of de novo design. Nature Communications, 8, 2263.

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Cryo-TEM Analysis of Nanoparticle-Enriched Extracellular Vesicles

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For cryo-TEM analysis, nPMVs and native EVs were concentrated (≥10¹² particles/mL) using Amicon Ultra-4 filters (10 kDa MWCO, 20-30 min, 4000 × g, 4 °C, Merck). Samples were adsorbed onto a carbon-coated grid (Lacey, Ted Pella, CA) and vitrified by rapid transfer into liquid ethane using a Leica GP plunger (Leica, Wetzlar, Germany). Frozen grids were transferred into a Talos electron microscope (FEI, Hillsboro, OR) using a Gatan 626 cryo-holder. Electron micrographs were recorded at an accelerating voltage of 200 kV and a nominal magnification of ×73,000, using a low-dose system (20 e⁻/Å²) and keeping the sample at low temperature.
For cryo-TEM tomography, samples were premixed with gold fiducials (10 nm) and adsorbed onto carbon-coated grids as described above. Tilt series were acquired from −60° to +60° in 3° steps in consecutive order. The total dose received by 61 images corresponds to ~200 e⁻/Å². Segmentation, generation of a nPMV density map, and sample visualization were performed^{125 (link)}. The final rendering of the nPMV model was created with Blender V3.3 (Blender Foundation, Amsterdam, Netherlands).

Alter C.L., Detampel P., Schefer R.B., Lotter C., Hauswirth P., Puligilla R.D., Weibel V.J., Schenk S.H., Heusermann W., Schürz M., Meisner-Kober N., Palivan C., Einfalt T, & Huwyler J. (2023). High efficiency preparation of monodisperse plasma membrane derived extracellular vesicles for therapeutic applications. Communications Biology, 6, 478.

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Cryo-EM Sample Vitrification and Imaging

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Cryo-electron microscopy (cryo-EM) was performed as described previously [29 ]. Briefly, samples were placed on a glow-discharged 300 mesh EM grid (Quantifoil R2/2) and vitrified using an EMGP (Leica, Germany) at room temperature and 100% humidity. Excess sample was removed by blotting for 1 s with a Whatman filter paper. The grids were plunged into liquid ethane (− 182 °C) and, following vitrification, the grid was stored in liquid nitrogen until further use. They were then mounted in a Gatan 626 cryo-holder for cryo-EM imaging using a Tecnai 12 electron microscope (FEI Company, the Netherlands) operated at 120 kV. The images were recorded on a 4 × 4 k Eagle camera (FEI Company, the Netherlands) at 18,000 × magnification (pixel size 1.2 nm) between 5 and 10 µm under focus.

Huis in ‘t Veld R.V., Lara P., Jager M.J., Koning R.I., Ossendorp F, & Cruz L.J. (2022). M1-derived extracellular vesicles enhance photodynamic therapy and promote immunological memory in preclinical models of colon cancer. Journal of Nanobiotechnology, 20, 252.

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Cryo-TEM Imaging of Peptide Assemblies

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Peptide solutions were assembled at concentrations of 5 mM and 20 mM for mfCMP-1a and mfCMP-2a, respectively, based on their solubility. mfCMP-1a rapidly formed large insoluble higher ordered structures with increased peptide concentration; however, no insoluble structures were observed in the case of mfCMP-2a at higher concentrations. After assembly, peptides were diluted to a concentration of 1 mM prior to sample preparation, mirroring the procedure used for preparation of mfCMP hydrogels. Lacey carbon film grids (200 mesh; Electron Microscopy Sciences, Hatfield, PA) were treated using a glow discharge plasma cleaner (PDC-32G, Harrica Plasma Inc., Ithica, NY) for 1 minute. The grids were loaded onto a Vitrobot, and 4 µL of assembled peptide solution was pipetted onto the grid, blotted twice for 2 seconds, and plunged into liquid ethane. The grids were transferred manually into holders immersed in liquid nitrogen and stored there until imaging. Imaging was performed at 200 kV on a Talos TEM with a Falcon 3 camera (FEI Company, Hillsboro, OR) using a Gatan 626 cryo holder at −176 °C. In the resulting images fibril widths and lengths were measured with FIJI.

Hilderbrand A.M., Ford E.M., Guo C., Sloppy J.D, & Kloxin A.M. (2019). Hierarchically structured hydrogels utilizing multifunctional assembling peptides for 3D cell culture. Biomaterials science, 8(5), 1256-1269.

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Cryo-EM Analysis of UC-MSC-EVs

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UC-MSC-EV samples were deposited on an electron microscopy (EM) grid coated with a perforated carbon film. Samples were quickly frozen in liquid nitrogen-cooled liquid ethane using a Leica EM-PC cryo system. EM grids were maintained under liquid nitrogen until use. EM grids transferred to a Tecnai F20 cryo-electron microscope (FEI, ThermoFisher) operating at 200 kV. Grids were mounted in a Gatan 626 cryo-holder and images were recorded with a FEI-Eagle camera.

Priglinger E., Strasser J., Buchroithner B., Weber F., Wolbank S., Auer D.P., Grasmann E., Arzt C., Narzt M., Grillari J., Preiner J., Jacak J., & Gimona M. (2020). Comprehensive label-free characterization of extracellular vesicles and their surface proteins.

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