For the analysis by electron microscopy, the P100 fractions were fixed with a solution of 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA), prepared in 0.2-M HEPES (pH 7.4) for 30 min (RT), followed by 10 min washes in acetone graded solutions (50 to 100%). Samples were then embedded in resin overnight (RT) and baked for 24 h at 65°C. Ultrathin sections (60 nm) were finally obtained and double stained with 2% uranyl acetate for 20 min and lead citrate for 10 min and collected on a nickel grid. Ten microliters of exosome suspension was used for negative staining. An antibody against CD63 (Santa Cruz Biotechnology, sc51662) and protein A gold (PAG 10 nm) were used for the immunostaining of exosomes. The grids were observed by transmission electron microscopy (TEM). EM images were acquired at 80 kW using an FEI Tecnai-12 electron microscope (ThermoFisher) equipped with a VELETTA CCD digital camera.
Fei tecnai 12 electron microscope
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The FEI-Tecnai 12 is a transmission electron microscope designed for high-resolution imaging. It features a tungsten filament as the electron source and operates at an accelerating voltage of 120 kV. The microscope is capable of magnifications ranging from 25x to 1,000,000x, allowing for detailed examination of specimen structures at the nanoscale level.
Automatically generated - may contain errors
Lab products found in correlation
Veleta, by EMSIS (2 mentions)
Veleta 4k ccd camera, by Olympus (2 mentions)
2k ccd veleta camera, by EMSIS (1 mentions)
Uranyl acetate, by Polysciences (1 mentions)
Epon 812, by Agar Scientific (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Osmium tetroxide, by Polysciences (1 mentions)
Glutaraldehyde, by Polysciences (1 mentions)
Em uc7, by Leica (1 mentions)
9 protocols using fei tecnai 12 electron microscope
1
Exosome Ultrastructural Analysis by TEM
2
Ultrastructural Tissue Fixation and Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos were fixed in 2% PFA, 2% glutaraldehyde in 0.1M cacodylate buffer, pH 7.4. Samples were embedded in 2% LMP-agarose and postfixed in 1% osmium tetroxide, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer. After that, the samples were dehydrated stepwise in ethanol, including en bloc 0.5% uranyl acetate staining during 70% ethanol. The last dehydration step was performed in propylene oxide followed by epon embedding. The samples were sectioned stepwise on an ultramicrotome (UC6, Leica) until the region of interest and 60-nm ultrathin sections were collected on a formvar-coated one-slot grid and counterstained with lead. The samples were imaged on a FEI-Tecnai 12-electron microscope at 80 kV (Thermo Fisher Scientific), and characteristic images were taken with a 2 K CCD-Veleta camera (EMSIS).
3
Ultrastructural Analysis of Mouse Hearts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hearts from freshly killed 12 week-old mice were removed, cut into halves and fixed in 2% paraformaldehyde, 2% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2. The left ventricle was cut in smaller cubes and subsequently post-fixed in 1% osmiumtetroxide, 1.5% potassiumferrocyanide in 0.1M cacodylate buffer, pH 7.2. The samples were stepwise dehydrated in ethanol, including en bloc 0.5% uranyl acetate staining during 70% ethanol incubation. Blocks were embedded in epon then sectioned ultrathin at 70 nm. Sections were collected on copper grids and stained with lead. The samples were analysed on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, The Netherlands) and representative areas were imaged with a 2 K CCD camera (Veleta, EMSIS, Münster, Germany).
4
Immunogold Labeling of Outer Membrane Vesicles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Electron microscopy of ultrathin cryosections of LB agar cultures of strains 5791/99 and 493/89, or of ultrathin cryosections of OptiPrep-purified 5791/99 OMVs was performed as described [10 (link)]. Briefly, overnight LB agar cultures harvested into PBS or a pool of OptipPrep fractions 1 to 8 of 5791/99 OMVs were fixed with 2% paraformaldehyde and 0.2% glutaraldehyde and processed using the method of Tokuyasu [85 (link)]. Ultrathin (50 nm) frozen sections were cut and immunogold-stained with anti-E. coli O157 LPS antibody (culture sections), or with anti-Stx2a, anti-CdtV-A, anti-CdtV-B, anti-EHEC-Hly, and anti-H7 antibody (OMV sections) and Protein A Gold (15 nm) (Department of Cell Biology, University Medical Center, Utrecht, The Netherlands). Staining with Protein A Gold alone, without first antibodies, served as a control of specificity of the immunogold staining. For negative staining, OMVs in OptiPrep fractions of 5791/99 and 493/89 OMVs were fixed and stained with 0.5% uranyl acetate. Samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, The Netherlands) and photographed (Ditabis imaging plates).
5
HPV16 Pseudovirus Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1-5x106 purified HPV16 or HPV16-L2(RTR313EEE) PsVs in PBS/0.8 M NaCl were absorbed for 1 min on formvar-coated, carbon-sputtered grids. Particles were contrasted for 4 min with 1% phosphotungstic acid, pH 7.2, and then for 10 s with 1% uranylacetate/ddH2O. Directly after drying, samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands). Photographs of selected areas were documented with Olympus Veleta 4k CCD camera.
6
Visualizing Cell-in-Cell Structures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EGFP-positive JAM-A KD MCF7 cells and LifeAct-mCherry-expressing MCF7 WT cells were mixed and grown on glass cover slides. Cell-in-cell structures were identified by confocal microscopy prior to fixation. Samples were subsequently fixed in 2.5% glutaraldehyde (Polysciences Europe GmbH, Hirschberg, Germany) in D-PBS (Sigma-Aldrich), post-fixed in 1% osmium tetroxide (Polysciences), block-stained with 0,5% uranyl acetate (Polysciences), dehydrated and flat-embedded in Epon 812 (Agar Scientific, Stansted, UK). Areas of interest were selected based on the fluorescence images and embedded in gelatine capsules (Polysciences). After polymerization, 60-nm ultrathin sections were counterstained with uranyl acetate (Polysciences) and lead (Pb(II) citrate, Sigma-Aldrich). Samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands). Images of selected areas were documented with ditabis imaging plates (Ditabis, Pforzheim, Germany).
7
Electron Microscopy of Mutant HPV16 PsVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mutant HPV16 PsVs were characterized by electron microscopy. Purified PsVs were diluted in virion buffer to a concentration of 1 × 108 particles/μl. PsVs were transferred on a Formvar-coated and carbon-spottered copper grid and sedimented for 10 min. Negative staining was performed by incubating sedimented PsVs with 1% phosphotungstic acid (pH 7.0) for 7 min and letting to dry. Samples were analyzed at 80 kV on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, Netherlands). Images were acquired with an Olympus Veleta 4k CCD camera.
8
Routine Transmission Electron Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For routine EM the cells were grown in 12 well plates as a monolayer. At the end of the experiment the cells were fixed with 1% Glutaraldehyde prepared in 0.2 M Hepes buffer. Then the cells were scraped, pelleted, post-fixed in OsO4 and uranyl acetate and embedded in Epon as described previously [43 (link)]. From each sample, thin 60 nm sections were cut using Leica EM UC7 (Leica Microsystems, Vienna, Austria). EM images were acquired from thin sections using a FEI Tecnai-12 electron microscope (FEI, Eindhoven, Netherlands) equipped with a VELETTA CCD digital camera (Soft Imaging Systems GmbH, Munster, Germany).
9
Ultrastructural Analysis of Embryo Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At the 2-cell stage, a total of 82 embryos (58 SN, 24 NSN) and 48 embryos (30 SN, 18 NSN) were collected after parthenogenesis and ICSI, respectively. They were fixed in 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for three hours at room temperature. Next the samples were adsorbed to poly-L-lysin coated cover slips.12 Subsequently samples were post-fixed in 1% osmium tetroxide, 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer, pH 7.2. Then they were stepwise dehydrated in ethanol, including en bloc 0.5% uranyl acetate staining during 70% ethanol. The coverslips were flat embedded in epon and polymerized. From the hardened sample the cover slip was removed and the epon bloc was ultrathin-sectioned at 70 nm. Sections were collected on copper grids and stained with lead. The samples were analyzed on a FEI-Tecnai 12 electron microscope (FEI, Eindhoven, The Netherlands) and imaged on a side-view camera (Veleta, EMSIS, Münster, Germany).
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!