In order to vitrify samples, a 2 µL droplet was put on a lacey carbon-filmed copper grid (Science Services, Munich, Germany) for 30 s [32 (link)]. Subsequently, blotting paper was used to remove most of the liquid, resulting in a thin film stretched over the lace holes. The specimens were instantly shock frozen by rapidly immersing them into liquid ethane cooled to approximately 90 K by liquid nitrogen in a temperature-controlled freezing unit (Zeiss Cryobox, Carl Zeiss Microscopy GmbH, Jena, Germany). All the steps of sample preparation were conducted at a controlled temperature. After specimen freezing, the remaining ethane was removed by blotting paper. The vitrified specimen was then transferred to a Zeiss/Leo EM922 Omega EFTEM (Zeiss Microscopy GmbH, Jena, Germany) transmission electron microscope using a cryoholder (CT3500, Gatan, Munich, Germany). The temperature of samples was maintained below 100 K during the examination. Specimens were examined with reduced doses of about 1000–2000 e/nm2 at 200 kV. Images were recorded by a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany) and analyzed using a GMS 1.9 software (Gatan, Munich, Germany).
Cryo box
Manufactured by Zeiss
Sourced in Germany
The Cryo-box is a laboratory equipment designed for the storage and transport of samples at cryogenic temperatures. It provides a secure and controlled environment to preserve the integrity of sensitive materials, such as biological samples or other temperature-dependent specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ultrascan 1000, by Ametek (4 mentions)
Ct3500, by Ametek (4 mentions)
Lacey carbon filmed copper grid, by Science Services (2 mentions)
Gms 1, by Ametek (2 mentions)
Leo em922 omega eftem, by Zeiss (2 mentions)
Leo 922 omega eftem, by Zeiss (1 mentions)
R1 2 1, by Quantifoil (1 mentions)
Gatan 626 dh, by Ametek (1 mentions)
Cm120, by Thermo Fisher Scientific (1 mentions)
Multi a holey carbon film, by Quantifoil (1 mentions)
Gatan 626 cryo transfer holder, by Ametek (1 mentions)
Emmanu4 v, by TVIPS (1 mentions)
7 protocols using cryo box
1
Vitrification of Samples for Electron Microscopy
2
Cryogenic Specimen Preparation for TEM
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Samples were vitrified as previously described [32 (link)]. Particularly a 2 µL sample droplet was put on a lacey carbon filmed copper grid (Science Services, Munich) for 30 s. Subsequently, a blotting paper has been employed to remove most of the liquid, resulting in a thin film stretched over the lace holes. The specimens were instantly shock frozen by rapid immersion into liquid ethane cooled to approximately 90 K by liquid nitrogen in a temperature-controlled freezing unit (Zeiss Cryobox, Carl Zeiss Microscopy GmbH, Jena, Germany). All the steps of sample preparation have been conducted at controlled temperature. After freezing the specimens, the remaining ethane has been removed by blotting paper. The vitrified specimen was transferred to a Zeiss/Leo EM922 Omega EFTEM (Zeiss Microscopy GmbH, Jena, Germany) transmission electron microscope using a cryoholder (CT3500, Gatan, Munich, Germany). Sample temperature was maintained below 100K during the examination. Specimens were examined with reduced doses of about 1000–2000 e/nm2 at 200 kV. Images have been recorded by a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany) and analyzed using a GMS 1.9 software (Gatan, Munich, Germany).
3
Cryo-TEM Analysis of Polymer Vesicles
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To examine the morphology and membrane thickness of polymer vesicles by transmission electron microscopy (TEM), a drop of the vesicle solution of interest was placed on copper grids with lacey carbon film (200 mesh, Plano, Wetzlar, Germany), blotted with filter paper, vitrified in liquid ethane (Zeiss Cryobox, Zeiss NTS GmbH, Germany) and stored in liquid nitrogen until inserted under nitrogen atmosphere within a cryo transfer holder (CT3500, Gatan, USA) to the TEM column. Specimens were observed at a temperature of approx. −180 °C using a LEO922 OMEGA EFTEM (Zeiss, Oberkochen, Germany) operated at 200 kV.
4
Cryogenic TEM Imaging of Samples
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A few microlitres of samples were mounted onto a copper TEM grid and the excess solution was removed with filter paper. The specimen was immersed into a cryo-box (Carl Zeiss NTS GmbH, filled with liquid ethane) to be rapidly cooled to −170 to −180 °C. The specimen was then transferred to a Zeiss EM922 EFTEM (Zeiss NTS GmbH, Oberkochen, Germany) by a cryo-transfer holder (CT3500, Gatan, Munich, Germany). TEM observation was made at −180 °C with an acceleration voltage of 200 kV. Reduced electron doses (500–2,000 e nm−2) were used to obtain zero-loss filtered images. A bottom-mounted CCD camera system (UltraScan 1,000, Gatan) was employed to record all images, which were then processed by Digital Micrograph 3.9.
5
Cryo-TEM Imaging of Proteorhodopsin
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Cryo-TEM was performed as described earlier (Rhiel et al. 2013 (link)). Briefly, a small droplet (5 μl) of the proteorhodopsin fraction, i.e., band B3 was placed on a copper grid covered by a holey carbon film (Quantifoil R 1.2/1.3, pore size 1.2 μm, 400 mesh; Quantifoil Micro Tools, Jena, Germany). Excess liquid was blotted for 3 s between two strips of filter paper. Subsequently, the sample was rapidly plunged into liquid ethane (cooled to about − 180 °C with liquid nitrogen) in a Cryobox (Zeiss, Oberkochen, Germany). The frozen specimen was transferred with a cryo-holder (Gatan 626-DH, Gatan, Pleasanton, USA) into a precooled cryo-transmission electron microscope (CM 120, FEI, Eindhoven, Netherlands) operated at 120 kV and viewed under low dose conditions. Cryo-TEM images were recorded with a 2 K CMOS Camera (F216, software EMMENU V4.0; camera and software TVIPS, Munich, Germany).
6
Cryogenic EM Imaging of Extracellular Vesicles
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EVs isolated from 4 × 104 infected monocytes (MOI 1:1) were collected in 4-µL suspension, and were applied onto copper EM-grids covered by a QUANTIFOIL Multi A holey carbon film (Quantifoil Micro Tools), and excess of liquid was blotted automatically between two strips of filter paper. Subsequently, the samples were rapidly plunge-frozen in liquid ethane (cooled by liquid nitrogen at about −180 °C) in a cryobox (Carl Zeiss). Excess of ethane was removed with a piece of filter paper. The samples were transferred immediately using a Gatan 626 cryo-transfer holder (Gatan) into a pre-cooled cryo-electron microscope (Philips) operated at 120 kV and imaged under low dose conditions. The images were recorded with a 2k F216-CMOS-camera (CMOS-camera and acquisition software, EMMANU4 v 4.00.9.17, TVIPS). In order to minimize the noise, four images were recorded and averaged to one image.
7
Cryogenic TEM Specimen Preparation
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A few microliters of sample was placed on a bare copper TEM grid (Plano, 600 mesh), and the excess liquid was removed with filter paper. This sample was cryo-fixed by rapidly immersing into liquid ethane cooled to À170 to À180 °C in a cryo-box (Carl Zeiss NTS GmbH). The specimen was inserted into a cryo-transfer holder (CT3500, Gatan, Munich, Germany) and transferred to a Zeiss EM922 EFTEM (Zeiss NTS GmbH, Oberkochen, Germany). Examinations were carried out at temperatures around À180 °C. The TEM was operated at an acceleration voltage of 200 kV. Zero-loss filtered images were taken under reduced dose conditions (500-2000 e/nm 2 ). All images were recorded digitally by a bottommounted CCD camera system (UltraScan 1000, Gatan) and processed with a digital imaging processing system (Digital Micrograph 3.9 for GMS 1.4, Gatan).
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