Electron microscopy experiments were executed as described in detail in Roybal et al. (2015b) (link). Briefly, Tg4WT or Tg4KO CD4+ T cells and peptide-loaded PL8s were centrifuged together for 30 s at 350 g to synchronize cell coupling, the cell pellet was immediately resuspended to minimize unspecific cell coupling and cellular deformation and the cell suspension was further incubated at 37 degree C. After 2 and 5 min for early and late time points, respectively, the cell suspension was high pressure frozen and freeze substituted to Epon. Ultrathin sections were analyzed in an FEI Tecnai12 BioTwin equipped with a bottom-mount 4*4K EAGLE CCD camera. T cell:APC couples were identified in electron micrographs through their wide cellular interface. As described in detail in Roybal et al. (2015b) (link), the time point assignment of cell couples was filtered with morphological criteria post acquisition using the presence of a uropod and T cell elongation.
Tecnai 12 biotwin
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands, United States
The Tecnai 12 BioTwin is a transmission electron microscope (TEM) designed for life science applications. It provides high-resolution imaging capabilities for the examination of biological samples. The Tecnai 12 BioTwin is capable of operating at accelerating voltages up to 120 kV, enabling detailed analysis of cellular structures and macromolecular complexes.
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Lab products found in correlation
Cf400 cu, by Electron Microscopy Sciences (4 mentions)
Nano z, by Malvern Panalytical (2 mentions)
Sis megaview 3, by Olympus (2 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Ckx53, by Olympus (1 mentions)
Cary 50, by Agilent Technologies (1 mentions)
Ti2 e a1 r, by Nikon (1 mentions)
Mcr 301, by Anton Paar (1 mentions)
Ultracut uct, by Thermo Fisher Scientific (1 mentions)
Fcf400 cu, by Electron Microscopy Sciences (1 mentions)
Xplore3d software, by Thermo Fisher Scientific (1 mentions)
Advantage hr hr b ccd camera system, by Advanced Microscopy Techniques (1 mentions)
Sigma vp, by Zeiss (1 mentions)
Dimension edge atomic force microscope, by Bruker (1 mentions)
Axs d8 advance, by Bruker (1 mentions)
Carbon coated grids, by Agar Scientific (1 mentions)
Leo em912 omega electron microscope, by Zeiss (1 mentions)
Illustrator cs5, by Adobe (1 mentions)
Ultracut uct microtome, by Leica (1 mentions)
Formvar coppergrids, by Ted Pella (1 mentions)
63 protocols using tecnai 12 biotwin
1
Ultrastructural Analysis of T Cell-APC Interactions
2
Bacteriophage Morphology by TEM
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Purified high titre bacteriophage was negatively stained with 2% (w/v) uranyl acetate. The sample was examined under FEI Tecnai 12 BioTwin transmission electron microscope at an operating voltage 100 kV. Detailed morphology that includes the shape and size of bacteriophage particles are studied.
3
Nanoparticle Characterization by DLS and TEM
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The resulted colloidal suspensions solubilized in PBS (sample S1) and in distilled water (samples S2 and S3) were characterized by dynamic light scattering—DLS, using a ZetaSizer NanoZS Malvern Instrument (Worcestershire, UK). The morphology and ultrastructure of aggregates and nanoparticles were characterized by transmission electron microscopy (TEM) using a FEI Tecnai 12 Biotwin, (Oregon, SUA) electron microscope. The samples obtained as described in the previous section were sonicated for 15 min. After the sonication, 1 drop from each sample was placed on the copper grid surface. This method was used to determine the size of the nanoparticles.
4
Comprehensive Nanoparticle Characterization
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UV-vis spectra were characterized by an UV-vis spectrophotometer (UV, Cary 50, Varian, Hong Kong). Transmission electron microscope (TEM, Tecnai-12 Bio-Twin, FEI, Netherlands) and scanning electron microscope (SEM, S-3400N, Hitachi, Japan) were performed to observe the nanoparticle morphology. Confocal laser scanning microscopy (CLSM, TI2-E+A1 R, Nikon, Japan) and digital microscope (CKX53, Olympus, Japan) were used to observe morphology of microneedles. The size distribution and zeta potential were determined by dynamic light scattering (DLS, ZS90, Malvern, UK). Flow cytometer (FCM, LSRFortessa, BD, USA) was performed to quantitatively analyze the immune cell phenotype.
5
Characterization and Stability of Anidulafungin Nanoparticles
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Anidulafungin NP particle size was determined by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern, Shanghai, China). The morphology was observed by transmission electron microscopy (TEM, Tecnai-12Bio-Twin, FEI, Netherlands). The effective encapsulation of AN into nanoparticles was confirmed by UV spectral analysis (Varian, Hong Kong, China). To evaluate stability, AN NP was incubated at either 4°C or room temperature for 30 days; alternatively, AN NP was also incubated at 37°C for 48 h. All measurements were conducted in triplicate. The solubility of 125 μg/mL AN with (experimental) or without (control) different ratios of rHSA in PBS was visually observed at 0 and 24 h. Two independent experiments were conducted.
6
Capsid-display Validation via EM
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Capsid-display of the pIX-modifications was confirmed with Electron Microscopy (EM). Purified pIX modified vectors were coated with the primary anti-CS antibody (clone 2A10) on a copper grid with a carbo-coated Formvar film and incubated for 60 minutes at room temperature (1:1). After washing, the grids were stained with gold labeled anti-mouse Protein A Gold 10 nm (PAG10) 1:200 in 1 mM PBS containing 2% bovine serum albumin (BSA) and 0.1% Tween 20 buffer for 60 minutes at room temperature and fixed using 1.5% glutaraldehyde in cacodylate buffer. Samples were subsequently negatively stained with 2% Silicotungstic acid (STA) and visualized with a transmission electron microscope (FEI Tecnai 12 BioTwin).
7
Electron Microscopy for Vessel Analysis
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For electron microscopy, mice were anesthetized using ketamine/xylazine, and the body circulation was perfused first with 10 ml PBS via a peristaltic pump, followed by 40 ml of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. The tissues were removed and post-fixed in reduced 1% osmium tetroxide containing 1.5% potassium hexacyanoferrate. Subsequently, samples were dehydrated and embedded in epon. 60-nm ultrathin sections were cut on an ultramicrotome (UC6; Leica), counterstained with uranyl and lead, and imaged on an electron microscope (Tecnai-12-biotwin; FEI). All vessels were analyzed in the area of the section (∼1 mm × 1 mm) and imaged for junction integrity (MegaView; Olympus). Furthermore, representative pictures of selected areas were imaged on high-resolution pictures with Ditabis plates.
8
Ultrastructural Analysis of Mouse Aorta
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For transmission electron microscopic analysis, mice were humanely killed by an anesthetic overdose with a ketamine/xylazine/acepromazine cocktail, followed by cardiac perfusion with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) (fixative). The aortae were then harvested and incubated in the fixative overnight. This was followed by a postfixation step with 1% osmium tetraoxide + 1.5% aqueous potassium ferrocyanide. Aortae were then dehydrated with an increasing concentration of acetone, followed by embedding in Epon. Ninety nanometer sections were transferred onto 200 mesh copper TEM grids and counterstained stained using 4% aqueous uranyl acetate and Reynold’s lead before imaging with an FEI Tecnai 12 BioTwin 120 kV transmission electron microscope.
9
Ultrastructural Analysis of Embryos and Larvae
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Embryos and larvae were fixed in 2% Glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 20 min at room temperature. Embryos were hand devitellinized. Both embryos and larvae were transferred in microcentrifuge tubes and fixed in 1% OsO4/2% Glutaraldehyde and then 2% OsO4. Specimens were washed and dehydrated in Araldite. Ultrathin sections of 0.1 µm were prepared and analyzed with Tecnai 12 BioTWIN (FEI Company).
10
Transmission Electron Microscopy of Cells
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After treatment, cells grown in chamber slides were fixed in 2.5% glutaraldehyde in PBS for one hour at room temperature, then stored at 4°C overnight before processing. Thin sections on grids were observed in a Tecnai 12 BioTwin transmission electron microscope (FEI) at 120 keV. Images were acquired with a charge coupled device camera (AMT).
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