Cell monolayers were fixed with a primary fixative consisting of 1% glutaraldehyde (Agar Scientific, Stansted, UK) at 4 °C over 3 days. After a primary fixation, the cell monolayers were washed and scraped off before being postfixed with 1% osmium tetroxide (Ted Pella, Redding, CA, USA) for 2 h. A few grains of potassium ferrocyanide were added to enhance the contrast of the membranous structure within the cells. After 2 h, the cell pellets were washed and dehydrated with progressively increased concentrations of ethanol (25%, 50%, 75%, 95% and 100%). The dehydration step was enhanced by another 2 rounds of absolute acetone treatment for 10 min each. Dehydrated cell pellets were then infiltrated by adding increasing concentrations of araldite 502 (Ted Pella) to acetone at increasing temperatures before being embedded in fresh araldite for 24 h at 60 °C. The embedded samples were then trimmed with an ultramicrotome (Reichert-Jung, Depew, NY, USA) to approximately 50 to 70 nm. Cut sections were then placed onto a 200-mesh copper grid before being stained with 2% uranyl acetate and postfixed with lead citrate. Stained sections were viewed using the Philips EM 208 transmission electron microscope (Philips, Eindhoven, The Netherlands) and captured digitally with a dual-view digital camera (Gatan Inc., Werrendale, CA, USA).
Em208 transmission electron microscope
Manufactured by Philips
Sourced in Netherlands
The EM208 is a transmission electron microscope (TEM) manufactured by Philips. It is designed to perform high-resolution imaging and analysis of various materials and biological samples at the nanoscale level. The EM208 utilizes a focused beam of electrons to interact with the sample, allowing for detailed visualization and examination of its internal structure and composition.
Automatically generated - may contain errors
Lab products found in correlation
Ultramicrotome, by Leica (2 mentions)
Glutaraldehyde, by Agar Scientific (1 mentions)
Osmium tetroxide, by Ted Pella (1 mentions)
Araldite 502, by Ted Pella (1 mentions)
Ultramicrotome, by Reichert Technologies (1 mentions)
Formvar coated copper grid, by Electron Microscopy Sciences (1 mentions)
0.1 m phosphate buffer, by Merck Group (1 mentions)
Uranyl acetate, by Electron Microscopy Sciences (1 mentions)
Parafilm, by Amcor (1 mentions)
Phosphate buffered saline (pbs), by Ted Pella (1 mentions)
Tristar 3000, by Micromeritics (1 mentions)
10 nm gold particles, by Merck Group (1 mentions)
Ls 55 luminescence spectrometer, by PerkinElmer (1 mentions)
Goat anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions)
Nicolet 370 ft ir spectrometer, by Thermo Fisher Scientific (1 mentions)
Mpms xl, by Quantum Design (1 mentions)
Epoxy resin, by Electron Microscopy Sciences (1 mentions)
Em uc6, by Leica (1 mentions)
15 protocols using em208 transmission electron microscope
1
Transmission Electron Microscopy of Cells
2
Transmission Electron Microscopy of EVs
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EVs were morphologically evaluated by transmission electron microscopy (TEM). Several drops of EV suspension (about 20 μL each) were placed on Parafilm (Bemis, Neenah, WI, USA). Formvar‐coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) were placed over them, in a moist chamber, for 30 minutes at room temperature. Grids were then washed once in 0.1 M phosphate buffer (Sigma‐Aldrich, St. Louis, MO, USA), pH 7.3, and twice in distilled water. Finally, they were contrasted with 2% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA), air dried, and observed under a Philips EM 208 transmission electron microscope equipped with a digital camera (University Centre for Electron Microscopy, CUME, Perugia, Italy).
3
Brain Ultrastructural Analysis of Mice
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The mice were sacrificed, as mentioned above. The brains were removed, and the SN was dissected and processed with 3% glutaraldehyde (Tianjin Hengxing Chemical Reagent Co., Ltd., Tianjin, China), 1.5% paraformaldehyde (Tianjin Hengxing Chemical Reagent Co., Ltd., Tianjin, China), and 0.1 M PBS (Sigma- Gibco, MO, United States, Cat# 70011069) at 4°C per 24 h each. This procedure was followed by post-fixations in 1% osmium tetroxide and 1.5% potassium ferrocyanide at 4°C for 1.5 h. After washing with PBS, the samples were dehydrated in a graded series of ethanol (75%, 95%, 100% v/v) and embedded in an Epon-Araldite solution (Ted Pella, Redding, CA, United States) at 60°C for 72 h. Then, the sections were cut at 100 nm thickness using an ultramicrotome (Leica, Wetzlar, Germany) and imaged under an EM208 transmission electron microscope (Philips, Eindhoven, the Netherlands).
4
Synthesis and Characterization of Halloysite Nanocomposites
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The chemical precursors and starting materials utilized in the synthesis process of samples, including halloysite nanotubes (HNTs), FeCl3·6H2O, Co(CH3CO2)2·4H2O, CTAB and NaOH, were purchased from a Merck company and applied without further purification. XRD diffractograms were recorded by an X-ray diffractometer device using Ni-filtered Cu Ka radiation (Philips-X’pertpro). FT-IR spectra were obtained on a Nicolet Magna-550 spectrometer in KBr pellets. SEM micrographs were obtained on a LEO-1455VP equipped with energy dispersive X-ray spectroscopy. EDS analysis with a 20 kV hasten voltage was performed. TEM images were captured on a Philips EM208 transmission electron microscope with an accelerating voltage of 200 kV. The BET analysis was conducted at − 196 °C using an automated gas adsorption analyzer (Tristar 3000, Micromeritics). The distribution of pore size was measured by applying the desorption branch of the isotherm in the BJH method.
5
Immunocapture and Visualization of GFLV
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Healthy, infected, or agro-infiltrated leaves were ground in 25 mM sodium phosphate buffer pH 7.4 and clarified at 4000 g for 5 min at 4°C. The 300 mesh formvar carbon-coated nickel grids were coated with polyclonal anti-GFLV antibodies and covered with clarified plant extracts. Following a saturation step with BSA 2% w/v, normal goat serum 10% v/v, Triton X100 0.05%, and 22.5 mM sodium phosphate buffer pH 7.4, the grids were further incubated with a mix of three homemade monoclonal anti-GFLV antibodies [17 (link)]. Immunogold labelling was performed using anti-mouse antibodies conjugated with 10 nm gold particles (Sigma-Aldrich, Saint-Louis, MO, USA). Three washes with sodium phosphate buffer preceded each step of the decoration procedure. Samples were negatively stained with a 1% ammonium molybdate solution prepared in the grinding buffer. For further analysis, a fixation step was performed before saturation, using a solution of 1% paraformaldehyde diluted in the grinding buffer. Observations were done on a Philips EM208 transmission electron microscope. Film-based photographs were acquired onto Kodak Electron Image Films SO-163 and developed. Photographs were scanned to obtain digital images.
6
Synthesis and Characterization of La2Sn2O7/g-C3N4 Nanocomposites
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All the chemical reagents for the synthesis of La2Sn2O7/ g-C3N4 nanocomposites such as La(NO3)3·6H2O (99.99%), SnCl4·5H2O (98%) and melamine were commercially available and employed without further purification. A multiwave ultrasonic generator (MPI Ultrasonics; welding, 1000 W, 20 KHz, Switzerland), immersed directly in the reaction solution. X-ray diffraction (XRD) patterns were recorded by a Philips-X’pertpro, X-ray diffractometer using Ni-filtered Cu Ka radiation. Fourier transform infrared (FT-IR) spectra were recorded on Nicolet Magna- 550 spectrometer in KBr pellets. The electronic spectrum of the sample was taken on Perkin–Elmer LS-55 luminescence spectrometer. Scanning electron microscopy (SEM) images were obtained on LEO-1455VP equipped with an energy dispersive X-ray spectroscopy. The EDX analysis with 20 kV accelerated voltage was done. Transmission electron microscopy (TEM) image was obtained on a Philips EM208 transmission electron microscope with an accelerating voltage of 200 kV.
7
Ultrastructural Analysis of Lung Tissues
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Lung tissues were fixed in TEM fixative (G1102, Servicebio, Wuhan, China) and post-fixed in 1% osmium tetroxide in the dark for 2 h at room temperature. The samples were rinsed in phosphate buffer (PB), dehydrated in ethanol, and embedded in resin. Ultrathin sections (60 to 80 nm) were cut on the ultramicrotome. Ultrathin sections were stained with 2% uranium acetate-saturated alcohol solution for 8 min, rinsed in 70% ethanol and ultra-pure water, stained with 2.6% lead citrate by avoiding CO2 for 8 min, and then rinsed with ultra-pure water. The samples were examined with an EM208 transmission electron microscope (Philips Electron Optics, Eindhoven, Netherlands).
8
Immunolabeling of Extracellular Vesicles for TEM Analysis
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Formvar-coated nickel grids with adherent EVs (see the previous section) were transferred for 10 minutes on single drops of 1% PBS-BSA (blocking buffer) to block unspecific sites. The grids were then incubated overnight at room temperature with mouse monoclonal anti-CD90 antibody (VMRD Inc., WA, USA) diluted 1 : 20 and rabbit anti-flotillin1 polyclonal antibody (Bioss Antibodies Inc., MA, USA) diluted 1 : 100 in blocking buffer. After several washes in PBS to remove the excess of antibody, the grids were incubated for 1 hour at room temperature, respectively, with goat anti-mouse secondary antibody and goat anti-rabbit, gold-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) diluted 1 : 40 in PBS with 1% BSA. Grids were finally washed in PBS, counterstained with 2% aqueous uranyl acetate for 5 min, washed with distilled water, and examined with a Philips EM 208 transmission electron microscope equipped with a digital camera (University Centre for Electron Microscopy (CUME) Perugia).
9
Characterization of Fe3O4@SiO2-phenyl-tetrazole
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All materials of commercial reagent grade were purchased from the Merck and Aldrich companies and used without further purification. FT-IR spectra were recorded on a Nicolet 370 FT/IR spectrometer (Thermo Nicolet, USA) using pressed KBr pellets. X-ray diffraction (XRD) measurements were carried out with a Philips powder diffractometer type PW 1373 goniometer. It was equipped with a graphite monochromator crystal. The X-ray wavelength was 1.5405 Å and the diffraction patterns were recorded in the 2θ range (10–80) with a scanning speed of 2° min−1. TEM images were taken using a Philips EM208 transmission electron microscope with an accelerating voltage of 90 kV. Scanning electron microscopy (SEM) of the {Fe3O4@SiO2@(CH2)35-phenyl-1H-tetrazole-SO3H/HCl} was performed on a Cam scan MV2300. The chemical compositions of the synthesized catalyst were determined by EDS (energy dispersive X-ray spectroscopy) performed in SEM. Thermal analysis (TG-DTG) was carried out using an STA 1500 Rheometric Scientific (England). The flow rate of air was 120 mL min−1 and the ramping rate of the sample was 2 °C min−1. VSM measurements were recorded using a SQUID magnetometer at 298 K (Quantum Design MPMS XL). Melting points were taken in open capillaries using BUCHI 510 melting point apparatus and are uncorrected.
10
Ultrastructural Analysis of Cardiac Mitochondria
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Hearts from perfused mice were further fixed in 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer over night at 4°C, post-fixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in epoxy resin (all from Electron Microscopy Science, Società Italiana Chimici, Rome, Italy). Semi-thin sections (2 µm) were stained with toluidine blue. Thin sections obtained with an MT-X ultratome (RCM, Tucson, AZ, USA) were mounted on copper grids, stained with lead citrate and examined with a EM208 transmission electron microscope (Philips, Eindhoven, The Netherlands). Mean mitochondrial area, mitochondria density (number of mitochondria/area expressed in µm2, normalyzed in 10 µm2) and number of mitochondria cristae (number of cristae/mitochondria area, normalized in 1 µm2) were determined by ImageJ.
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