PLA, BODIPY® 500/510 C4, C9 (0.2%, ThermoFisher Scientific, Villebon sur Yvette, France) (5-Butyl-4,4-Difluoro-4-Bora-3a,4a-Diaza-s-Indacene-3-Nonanoic Acid) and Coumarin 6 (0.2%; Sigma, Saint-Quentin-Fallavier, France) PLA nanoparticle batches were provided by Adjuvatis (Lyon, France). Blank and fluorescent modified PLA nanoparticles were made by nanoprecipitation, as previously described [24 –26 (link)]. PLA nanoparticle batches were characterized for their hydrodynamic diameter and their zeta potential in PBS solution using a Zetasizer NanoZS (Malvern, Orsay, France) at 25°C. Negative staining for transmission electron microscopy (TEM) analysis was done for PLA nanoparticles coated with different concentrations of RGDS-protein as previously described [27 ]. TEM observations were done with a Philips CM120 transmission electron microscope at the "Centre Technologique des Microstructures" (Lyon, France), and images were captured using a Gatan Orius 200 2Kx2K Camera. Particle size distribution was determined using Image J software.
Cm120 transmission electron microscope
Manufactured by Ametek
Sourced in France
The CM120 is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of microscopic samples. It features a 120 kV electron beam and advanced optics for achieving nanometer-scale resolution. The CM120 provides researchers and scientists with a powerful tool for in-depth study of materials at the atomic and molecular level.
Automatically generated - may contain errors
Lab products found in correlation
Orius ccd camera, by Ametek (2 mentions)
Bodipy 500 510 c4 c9, by Thermo Fisher Scientific (1 mentions)
Orius 200 2kx2k camera, by Ametek (1 mentions)
Nano z, by Malvern Panalytical (1 mentions)
Coumarin 6, by Merck Group (1 mentions)
Gatan ccd camera, by Ametek (1 mentions)
Uc7 microtome, by Leica camera (1 mentions)
Reichert ultrostainer, by Leica camera (1 mentions)
Orius sc200 ccd camera, by Ametek (1 mentions)
0.1 m sodium cacodylate buffer, by Agar Scientific (1 mentions)
Usc1000 ssccd camera, by Ametek (1 mentions)
Em uc6 ultramicrotome, by Leica camera (1 mentions)
Osmium tetroxide, by Agar Scientific (1 mentions)
6 protocols using cm120 transmission electron microscope
1
Characterization of Fluorescent PLA Nanoparticles
2
High-Pressure Freeze Substitution Electron Microscopy
3
Exosome Visualization by TEM Imaging
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For transmission electron microscopy (TEM), purified exosomes from cells were re-suspended in PBS and imaged as detailed by Jia et al. [24] (link). Briefly, EV samples were added onto formvar coated copper grids for 2-min, then washed in ultrapure water and negatively stained with 2% phosphotungstic acid (PTA). The samples were visualised using a Phillip CM120 transmission electron microscope (Eindhoven, The Netherlands) and photographed by a Gatan CCD camera (Gatan, Pleasanton, CA).
4
High-Pressure Freeze Substitution Electron Microscopy
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For sample preparation, 3 ml of culture from meiotic cell cycle time-course was vacuum filtered through a 0.45 μm Millipore filter. The cell paste was rapidly frozen under high pressure in a Wohlwend Compact 02 High Pressure Freezer. Frozen cell pellets were then freeze substituted in acetone containing 2% (w/v) osmium tetroxide and 0.1% (w/v) uranyl acetate at -80°C. Samples were slowly warmed to room temperature over three days. After washing cells twice in acetone, samples were embedded in Epon 812 resin (Hexion) through multiple changes of diluted resin with acetone (1:3, 1:1 and 3:1). Three more changes using undiluted Epon 812 resin were carried out over two days before resin was polymerised at 60–70°C overnight. Epon blocks were serially sectioned at a thickness of 70 nm and stained with 2% (w/v) uranyl acetate in sterile water for 8 mins, and then in Reynolds’ lead citrate for 3 mins. Sections were viewed on a Philips CM120 transmission electron microscope, and images were collected with a Gatan Orius CCD camera and processed using ImageJ v1.47.
5
Microscopic Analysis of Bacterial Mutants
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Wild type and glnR-codY- (fecE-) cells were exponentially grown at 37°C in C+Y medium. Samples were then collected, centrifuged and fixed overnight with 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5) at 4°C. Postfixation with 1% osmium tetroxide in cacodylate buffer was carried out for 1 h at room temperature. These fixed cells were dehydrated using a graded series of ethanol and embedded in LR White at 60°C for 48 h. Ultrathin sections (50 nm) were obtained using a Leica UC7 microtome and were counterstained with uranyl acetate and lead citrate (Reichert Ultrostainer, Leica, Germany). Samples were examined with a Philips CM120 transmission electron microscope equipped with a Gatan Orius SC200 CCD camera.
6
Transmission Electron Microscopy of Shear-Stressed Cells
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5 × 105 cells submitted or not to shear stress assay were centrifuged at 200g for 5 min in a 1.5 mL polypropylene tube. The pellet was incubated with a solution of Karnovsky fixative at 4 °C overnight. The pellet was washed with 0.1 M sodium cacodylate buffer (Agar Scientific, Stansted, UK), then post-fixed for 1 h in 1% osmium tetroxide (Agar Scientific) in sodium cacodylate buffer and then washed three times and finally re-suspended in 20 µL of cacodylate buffer. A 5 µL drop was introduced into the core of a warm (40–50 °C) 8% agarose fluid gel in a 1.5 mL polypropylene tube. Once solidified, a 3 mm3 block was cut around the drop. Dehydration process was performed by 3 successive baths of 10 min in 100% ethanol followed by incubation in propylene oxide. The sample was then embedded in Epon-Araldite and ultra-thin sections (65 nm with Leica EM-UC6 ultra-microtome) were stained for 10 min in 5% uranyl acetate and 5 min in lead citrate. The sections were imaged with a FEI CM120 transmission electron microscope at 120 kV, using a Gatan USC1000-SSCCD camera.
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