Purified concentrated EVs in PBS were mixed with an equal volume of 4% paraformaldehyde (PFA). Five microliters of resuspended pellets were added to a formvar-carbon-coated EM grid and absorbed for 20 min in a dry environment. The grids were washed once with PBS for 1 min, followed by once with 1% glutaraldehyde for 5 min, and seven times with Milli-Q water for 2 min per wash. The grids were then negatively stained with 2.5% uranyl-oxalate solution for 10 min and air-dried for 5 min under incandescent light. Images were acquired on a Tecnai G2 Spirit TEM (FEI, The Netherlands) with a wide-angle AMT 2k CCD camera operating at 120 kV at the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.
Amt 2k ccd camera
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AMT 2k CCD camera is a scientific imaging device designed for laboratory applications. It features a 2048 x 2048 pixel sensor that can capture high-resolution images. The camera is capable of operating at various frame rates and bit depths to suit different imaging requirements.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick reagent, by System Biosciences (3 mentions)
Tecnai g2 spirit biotwin tem, by Thermo Fisher Scientific (2 mentions)
Tecnai g2 spirit tem, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit twin transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
Nanoparticle tracking analysis, by Particle Metrix (1 mentions)
Tecnai g2 spirit biotwin instrument, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 spirit biotwin, by Thermo Fisher Scientific (1 mentions)
Transwell clear, by Corning (1 mentions)
Tecnai g spirit biotwin transmission electron microscope, by Thermo Fisher Scientific (1 mentions)
9 protocols using amt 2k ccd camera
1
Negative Staining of Extracellular Vesicles
2
Transmission Electron Microscopy of MSC-MVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSC-MVs purified by ultracentrifugation were applied to carbon-coated grids and stained with 1% uranyl acetate. The grids were examined by Tecnai G2 Spirit TWIN Transmission electron microscope (FEI) at an acceleration voltage of 80 kV. Photographs were taken with an AMT 2k CCD camera.
3
Isolation and Characterization of CSF Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To isolate CSF EVs, CSF samples (250 μl) were centrifuged at 3000g to remove cellular debris and supernatants were incubated overnight at 4°C with ExoQuick reagent (System Biosciences, Inc., Mountain View, California, USA) according to manufacturer's instructions. The suspensions were centrifuged at 1500g for 30 min and CSF EV pellets were suspended in 20 μl PBS for transmission electron microscopy (TEM) or RIPA buffer (Triton X-100 1%, NaCl 150 mmol/l, sodium deoxycholate 0.5%, Tris-HCL 50 mmol/l, SDS 0.1%, pH 7.4) for ELISA and western blotting. The supernatants (EV-depleted CSF) were stored at −80°C. EV concentrations and sizes were measured by nanoparticle tracking analysis (NTA) (Particle Metrix, Germany) either directly from CSF samples (diluted 1 : 500) or from isolated EV fractions (diluted 1 : 5000). TEM was performed using a Tecnai G2 Spirit BioTWIN instrument (FEI company, Hillsboro, Oregon, USA) equipped with an AMT 2k CCD camera (Harvard University TEM core).
4
Ultrastructural Analysis of Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown on Transwell-Clear (Corning) were processed for EM as described previously (Tang, 2006 (link)). In brief, MDCK and T84 epithelial cells grown on Transwells were chilled at 4°C for 6 h before fixation with 3.75% glutaraldehyde, 150 mM NaCl, and 20 mM Hepes, pH 7.5, at 4°C for 18 h. The fixation reaction was quenched with 50 mM glycine and 150 mM Hepes, pH 7.5, on ice for 1 h. Cells/Transwells were rinsed in ice-cold distilled water three times, secondarily fixed with 1% osmium tetroxide/1.5% potassium ferrocyanide for 2 h on ice, rinsed four times in ice-cold distilled water, en bloc stained with freshly prepared and filtered 2% uranyl acetate in distilled water on ice for 2 h, and rinsed four times in ice-cold distilled water. Cells/Transwells were dehydrated with sequential 5-min incubations in 50%, 75%, 95%, 100%, 100%, and 100% ethanol at room temperature. epon-Araldite (EMbed 812) was added to Transwells and allowed to polymerize at 60°C for 48 h. Ultrathin sections were cut using an Ultracut-S microtome (Reichert), layered onto carbon-coated copper grids, and stained with freshly made/filtered 2% lead citrate. Grids were examined with Tecnai G2 Spirit BioTwin (FEI) equipped with an AMT 2K CCD camera. Digital images acquired were imported into Photoshop for figure preparation.
5
Electron Microscopy of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One drop (5 μL) of a sample containing EVs was floated on the grid storage box for 1 min. The grid was then moved to a drop of double-distilled water, and the excess liquid was removed with a filter paper and stained by floating on a small drop of uranyl formate 0.75% for 30 s. After removing the excess uranyl formate with a filter paper, the grids were examined in a TecnaiG2 Spirit BioTWIN TEM (FEI Company, Hillsboro, OR, USA), and images were recorded with an AMT 2k CCD camera at a primary magnification of × 20,000–50,000 (Harvard Medical School’s Electron Microscopy Core Facility).
6
TEM Imaging of Extracellular Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A single drop (5 μL) of isolated EV sample was placed on a copper grid for 1 min. The grid was then washed with a drop of DEPC water, and the excess liquid was removed with a Whatman paper. The grids were stained by incubating with 2 μL of uranyl formate 0.75% for 30 s. After removing the excess uranyl formate in a similar manner, the grids were scanned with a TecnaiG2 Spirit BioTWIN TEM (FEI, Hillsboro, OR, USA), and images were taken with an AMT 2k CCD camera at a magnification of 18,500–30,000×.
7
Exosome Characterization in Rheumatoid Arthritis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Exosomes were extracted from serum cells of RA patients and controls, using the ultracentrifugation method [29 (link)] and exosome extraction/purification kit (Thermo Fisher Scientific, CA, USA). The extracted exosomes were later characterized, using dynamic light scattering (DLS) and transmission electron microscopy (TEM) [29 (link)]. In TEM analysis, 5μl of exosomes sample was absorbed for 1 minute on carbon-coated grid. Exosomes were then stained with 0.75% uranyl formate for 20–30 seconds.
After removing the excess stain with filter paper, the grids were examined in a transmission electron microscope (JEOL 1200EX, USA) and images were recorded with an AMT 2k CCD camera (Thermo Fisher, USA).
Furthermore, exosomes extracted from serum samples of patients and controls were confirmed by CD9, CD63, and CD81 antibodies. This confirmation was performed by commercially available CD9, CD63, and CD81 ELISA kits (Abcam). Absorbance was estimated using a microplate reader (Platos R496, AMP Diagnostics), in which the calibration curves were obtained indicating the accurate sample calibrations.
After removing the excess stain with filter paper, the grids were examined in a transmission electron microscope (JEOL 1200EX, USA) and images were recorded with an AMT 2k CCD camera (Thermo Fisher, USA).
Furthermore, exosomes extracted from serum samples of patients and controls were confirmed by CD9, CD63, and CD81 antibodies. This confirmation was performed by commercially available CD9, CD63, and CD81 ELISA kits (Abcam). Absorbance was estimated using a microplate reader (Platos R496, AMP Diagnostics), in which the calibration curves were obtained indicating the accurate sample calibrations.
8
Negative Staining of DREP4 CIDE Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DREP4 CIDE samples in 50 mM NaCl or 2 M NaCl, after purification by affinity chromatography, were diluted to 0.5 mg/mL. For negative staining, 10 μL of each protein sample was placed on a glow-discharged copper grid and stained with 1% uranyl formate at pH 4.5 for 30 s, and air-dried. The grids were imaged using the Tecnai G² Spirit BioTWIN Transmission Electron Microscope and recorded with an AMT 2k CCD camera (Thermo Fisher Scientific, Sunnyvale, CA, USA).
9
Negative Staining of DREP2 and DREP4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Wild-type DREP2 CIDE and R36E mutant samples after affinity chromatography purification were diluted to 0.8 mg/mL to prepare the high concentration sample and 0.1 mg/mL to prepare the low concentration sample. DREP4 CIDE samples were also diluted to 1 mg/mL as the high concentration sample and 0.1 mg/mL as the low concentration sample. For negative staining, 10 μL of each protein sample was placed onto a glow discharged copper grid and stained with 1% uranyl formate at pH 4.5 for 30 s and air-dried. The grids were imaged using a Tecnai G² Spirit BioTWIN Transmission Electron Microscope (FEI Company, Hillsboro, OR, USA) and recorded with an AMT 2k CCD camera (Thermo Fisher Scientific, Waltham, MA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!