The presence of oligomeric forms of Aβ42 in the culture supernatants was additionally assessed by electron microscopy using our previously described protocols [8 (link), 14 (link)]. Briefly, 3 μl aliquots were placed onto carbon-coated 400-mesh Cu/Rh grids (Ted Pella, Inc., Redding, CA, USA) and stained with 1% uranyl acetate in distilled water (Polysciences, Inc., Warrington, PA, USA). Stained grids were examined in a Philips CM-12 transmission electron microscope and photographed with a Gatan (4 k × 4 k) digital camera at the NYU School of Medicine Microscopy Laboratory Core Facility.
Cm 12 transmission electron microscope
Manufactured by Ametek
Sourced in United Kingdom
The CM-12 is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of samples. It features a high-intensity electron beam and advanced optics for detailed observation of micro- and nano-scale structures. The CM-12 is capable of magnifications up to 1,000,000x and provides detailed information about a sample's composition and morphology.
Automatically generated - may contain errors
Lab products found in correlation
Uranyl acetate, by Polysciences (3 mentions)
Carbon coated 400 mesh cu rh grid, by Ted Pella (2 mentions)
Gatan 4 k 2.7 k digital camera, by Ametek (2 mentions)
Cu rh grid, by Ted Pella (1 mentions)
Ultrostainer, by Philips (1 mentions)
Formvar coated grids, by Agar Scientific (1 mentions)
Cacodylate buffer, by Electron Microscopy Sciences (1 mentions)
Orius 1000 ccd camera, by Ametek (1 mentions)
K3fe cn 6, by Merck Group (1 mentions)
Oneview camera, by Ametek (1 mentions)
Histopaque, by Merck Group (1 mentions)
7 protocols using cm 12 transmission electron microscope
1
Oligomeric Aβ42 Visualization by Electron Microscopy
2
Amyloid Homologue Structure Analysis
3
Cryo-TEM Characterization of Nanomedicines
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The shape and morphology of the formulated reverse-micelles, liposomes, and iontosomes were studied using transmission electron microscopy at cryogenic temperature (Cryo-TEM) using a previously reported protocol [24 (link)]. Briefly, 5 μL of each formulation was applied to a copper grid pre-coated with perforated carbon film. After blotting the excess formulation, the grid was immediately immersed in liquid ethane container cooled using liquid nitrogen. Cryo-TEM images were recorded at −170 °C using a Philips CM12 transmission electron microscope (Eindhoven, The Netherlands) operating at 100 kV and equipped with a cryo-specimen holder Gatan 626 (Warrendale, PA, USA). Digital images were recorded with a Gatan MultiScan CCD camera and processed using the Gatan Digital Micrograph.
4
Ultrastructural Analysis of Myelinated Nerve Fibers
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Intercostal nerves were incubated for 48 h in 4% PFA: 2.5% glutaraldehyde at 4°C before post-fixation in 1% osmium tetroxide in 0.1 M phosphate buffer for 45 min. Following dehydration through an ascending series of ethanol solutions and propylene oxide, sections were embedded on glass slides in Durcupan resin. Regions to be used for the assessment of myelination were then cut out from a randomly selected section using a scalpel and glued onto a resin block for sectioning. Ultrathin sections (60 nm) were cut and collected on formvar-coated grids (Agar Scientific, UK), stained with uranyl acetate and lead citrate in an LKB Ultrostainer and then quantitatively assessed in a Philips CM12 transmission electron microscope equipped with a Gatan digital camera. Intercostal nerve fibres were measured using ImageJ. For each individual fibre, axon diameters and G-ratios were calculated as previously described (46 (link)).
5
Electron Microscopy of Amyloid-Beta Peptide Oligomerization
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Peptide oligomerization was also monitored by electron microscopy, as previously described [38 (link), 66 (link), 71 (link)]. Five microliters of either monomeric or oligomeric preparations of the different Aβ peptides were placed onto carbon-coated 400 mesh Cu/Rh grids (Ted Pella, Inc., Redding, CA) and stained with 1% uranyl acetate in distilled water (Polysciences, Inc., Warrington, PA). The stained grids were examined in a Philips CM-12 transmission electron microscope and photographed with a Gatan (4 k × 2.7 k) digital camera at the Image Core Facility of the Skirball Institute of Biomedical Medicine, NYU School of Medicine, as described [66 (link), 71 (link), 72 (link)].
6
Ultrastructural Analysis of Cells
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Cells were fixed with 2.5% glutaraldehyde/2.0% paraformaldehyde (PFA) (Electron Microscopy Sciences) in 0.1M Cacodylate buffer at room temperature for 2 hr, then rinsed in 0.1M Cacodylate buffer (Electron Microscopy Sciences) and post-fixed with 1% OsO4 (Electron Microscopy Sciences) and 0.8% K3Fe(CN)6 (Sigma-Aldrich) for 1 hr at 4°C. Then, samples were rinsed in 0.1M Cacodylate buffer followed by a water rinse and stained with 1% uranyl acetate, overnight at 4°C. The samples were stepwise dehydrated in Ethanol (50%, 70% 2 × 10 min, 95% 2 × 15 min and 100% 3 × 15 min), infiltrated in a graded series of Epon (Ethanol100%/Epon 3/1, 1/1, 1 hr) and kept in Ethanol100%/Epon 1/3 overnight. The following day, samples were placed in pure Epon and polymerized at 60°C. One hundred nm thin sections were collected in 200 copper mesh grids and imaged with a Philips CM12 transmission electron microscope operated at 80 kV and equipped with an Orius 1000 CCD camera (Gatan).
7
Pancreatic Islet Isolation Protocol
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Detailed methods are provided in the online version of this paper and include the following: supernatant was removed and the tissue was re-suspended in 4 ml Histopaque (Sigma 11191) in a 15 ml falcon tube. To the same tube, 4 ml of Histopaque (Sigma 10771) was added dropwise followed by 3 ml of RPMI medium. Centrifugation was set to 2500 rpm at 4 C without brakes and acceleration for 20 min to separate pancreatic islets from the exocrine tissue. Islets were picked with a Pasteur pipette and washed twice with 10 ml RPMI 1640 complete medium (11mM glucose, 10% FBS, 1% penicilin/streptomycin, and 1% L-glutamine). Islets were handpicked under the binocular. S5B andS5C). Figure S5A, images were recorded using a Philips CM-12 transmission electron microscope equipped with a Gatan OneView camera. Serial sections were stitched with the Software SerialEM (Mastronarde, 2005) . To quantify the fraction of putative extreme cells in the TEM images we included all beta cells with an apparent nucleus in the sectioned slice (224 cells). Extreme cells were identified as cells with an expanded ER close to the cell border and low insulin granule counts.
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