The DNA template for CpMgm101 binding was a 738-bp EcoRI fragment derived from the mitochondrial telomere of the C. parapsilosis linear mitochondrial DNA. The fragment was prepared by digestion of the pTZ19R-E1 plasmid (2 (link)) with EcoRI followed by a 1 min incubation with exonuclease III to generate single-stranded overhangs at the 5′ termini. CpMgm101 and this DNA were mixed in a 10:1 ratio (w/w) and incubated for 10 min at 30°C in a buffer B. Immediately after incubation they were diluted in spraying buffer (100 mM ammonium acetate, 30% (v/v) glycerol, pH adjusted to 8.0 with NaOH) to a final concentration of 225 μg/ml CpMgm101 and 20 μg/ml DNA. The diluted samples were sprayed onto freshly cleaved mica chips and transferred into a BalTec MED020 high vacuum evaporator equipped with electron guns. The rotating samples were coated with 0.7 nm platinum at an angle of 7°, followed by 7 nm carbon at 45°. The produced replicas were floated off from the mica chips and picked up with 400 mesh Cu/Pd grids. The grids were inspected in an FEI Morgagni 268D TEM operated at 80 kV. Images were acquired using an 11 megapixel Olympus-SIS Morada CCD camera.
Morgagni 268d tem
Manufactured by Thermo Fisher Scientific
Sourced in United States, Netherlands
The Morgagni 268D TEM is a transmission electron microscope (TEM) manufactured by Thermo Fisher Scientific. It is designed for high-resolution imaging and analysis of a wide range of materials and samples. The Morgagni 268D TEM provides the core function of magnifying and projecting an image of a thin specimen onto a fluorescent screen or digital detector for observation and analysis.
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Lab products found in correlation
Morada ccd camera, by Olympus (8 mentions)
Mica chips, by Agar Scientific (3 mentions)
Cu pd grids, by Agar Scientific (3 mentions)
Uranyl acetate, by Merck Group (2 mentions)
Sis morada ccd camera, by Olympus (1 mentions)
Ab6556, by Abcam (1 mentions)
Ultracut emfcs ultramicrotome, by Leica (1 mentions)
Embed 812, by Electron Microscopy Sciences (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Megaview camera, by Olympus (1 mentions)
Lambda 750s uv vis nir spectrometer, by PerkinElmer (1 mentions)
Sta 6000, by PerkinElmer (1 mentions)
Iraffinity 1, by Shimadzu (1 mentions)
Has 3000, by Malvern Panalytical (1 mentions)
21 protocols using morgagni 268d tem
1
Visualizing CpMgm101 Binding to mtDNA
2
Immunolabeling of GFP-tagged Proteins
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Prior to immunolabeling, grids were placed on plates with solidified 2% gelatine and warmed up to 37°C for 20 min to remove the pick-up solution. After quenching of free aldehyde-groups with glycine (0.1% for 15 min), a blocking step with 1% BSA (fraction V) in 0.1 M Sörensen phosphate buffer (pH 7.4) was performed for 40 min. The grids were incubated in primary antibody, rabbit polyclonal to GFP (ab6556, Abcam, UK), diluted 1:125 in 0.1 M Sörensen phosphate buffer over night at 4°C, followed by a 2 hr incubation in the secondary antibody, a goat-anti-rabbit antibody coupled with 6 nm gold (GAR 6 nm, Aurion, The Netherlands), diluted 1:20 in 0.1 M Sörensen phosphate buffer, performed at RT. The sections were stained with 4% uranyl acetate (Merck, Germany) and 2% methylcellulose at a ratio of 1:9 (on ice). All labeling steps were conducted in a wet chamber. The sections were inspected using a FEI Morgagni 268D TEM (FEI, The Netherlands) operated at 80kV. Electron micrographs were acquired using an 11-megapixel Morada CCD camera from Olympus-SIS (Germany).
3
Assessing 16x NCP Array Quality
4
Cohesin Complexes Analyzed by EM
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Cohesin was diluted to a concentration of approximately 0.1 mg/mL in FLAG buffer in the absence or presence of 1 μM rapamycin. Hinge domains were diluted to 0.1 mg/ml in 20 mM Tris pH 7.5, 100 mM NaCl. Samples were subsequently diluted 1:1 in spraying buffer, containing 200 mM ammonium acetate and 60% (v/v) glycerol, pH adjusted to 7.6.
After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with 0.7 nm Platinum (BALTIC, Germany) at an angle of 4-5°, followed by 8 nm Carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and inspected in an FEI Morgagni 268D TEM (FEI, the Netherlands) operated at 80 kV. Positions on the grid were chosen randomly. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with 0.7 nm Platinum (BALTIC, Germany) at an angle of 4-5°, followed by 8 nm Carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and inspected in an FEI Morgagni 268D TEM (FEI, the Netherlands) operated at 80 kV. Positions on the grid were chosen randomly. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
5
Ultrastructural Analysis of PBMC Mitochondria
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PBMCs were washed three-times in PBS and fixed with 2% glutaraldehyde in PBS for 2 h at 4 °C. Samples were post-fixed with 1% osmium tetroxide in veronal acetate buffer pH 7.4 for 1 h at 25 °C, stained with uranyl acetate (5 mg/ml) for 1 h at 25 °C, dehydrated in acetone and embedded in Epon 812 (EMbed 812, Electron Microscopy Science, Hatfield, PA, USA). Ultrathin sections obtained with an Ultracut EMFCS ultramicrotome (Leica Microsystems, Wetzlar, Germany) were unstained or poststained with uranyl acetate and examined under a Morgagni 268D TEM (FEI, Hillsboro, OR, USA) equipped with a Mega View II charge-coupled device camera (SIS, Soft Imaging System GmbH, Munster, Germany).
For the two-dimensional morphometric analysis of mitochondria, at least 20 cell sections in ten different microscopic fields were randomly captured from ultrathin sections of each PBMCs sample and digitalized at 28,000X original magnification. Area of all mitochondria and single cell sections were measured with the AnalySIS software (Soft Imaging System).
For the two-dimensional morphometric analysis of mitochondria, at least 20 cell sections in ten different microscopic fields were randomly captured from ultrathin sections of each PBMCs sample and digitalized at 28,000X original magnification. Area of all mitochondria and single cell sections were measured with the AnalySIS software (Soft Imaging System).
6
SUMO Chain Synthesis Visualization
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Reaction products of an in vitro SUMO chain synthesis were diluted to a final concentration of 50 µg/ml in spraying buffer, containing 100 mM ammonium acetate and 30% (v/v) glycerol, pH adjusted to 7.4. After dilution, the samples were sprayed onto freshly cleaved mica chips (Christine Gröpl, Austria) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. The rotating samples were coated with 0.6 nm Platinum (BALTIC, Germany) at an angle of 4–5°, followed by 8 nm carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, U.K.) and inspected in an FEI Morgagni 268D TEM (FEI, The Netherlands) operated at 80 kV. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
7
Visualizing capFFV nanoparticles by TEM
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TEM samples from capFFV at 0.6, 1, and 2% w/v were prepared using Formvar-coated copper TEM grids (400 mesh). Grids were prepared by glow discharge as described by Medini et al. [14 (link)]. TEM micrographs were taken using a Tecnai F20 (FEI Company, Oregon, USA) operating at 200 kV and a Morgagni 268D TEM (FEI Company, Oregon, USA) operating at 80 kV.
8
Nanoparticle Size and Morphology Analysis
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The size and size distribution of the nanoparticles were measured by Zetasizer Nano ZS, (Malvern Instruments Ltd., Worcestershire, UK). One mL of sample was used for the analysis. Since all the particles in the medium exhibit Brownian motion, scattering of light will occur, and the changes in intensity of light will detect suitable optics and a photo multiplier.
Transmission electron microscopy (TEM) was used for further evaluation of the particle size and surface morphology for the optimized nanoparticles (Morgagni 268D TEM, FEI, Hillsboro, OR, USA) at an acceleration voltage of 200 kV and viewed at a magnification of 50,000× g. A drop of the diluted sample of the nanoparticle was placed on a copper grid and stained with phosphotungstic acid, and after complete drying, the sample was analyzed, and images were captured [31 (link)].
Transmission electron microscopy (TEM) was used for further evaluation of the particle size and surface morphology for the optimized nanoparticles (Morgagni 268D TEM, FEI, Hillsboro, OR, USA) at an acceleration voltage of 200 kV and viewed at a magnification of 50,000× g. A drop of the diluted sample of the nanoparticle was placed on a copper grid and stained with phosphotungstic acid, and after complete drying, the sample was analyzed, and images were captured [31 (link)].
9
Nanoemulsion Characterization and Cytotoxicity
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Particle size, polydispersity and z-potential of nanoemulsions were determined by dynamic-light-scattering (DLS) and phase-analysis light scattering (PALS) according to Artiga-Artigas et al. [18 (link)]. To avoid multiple scattering effects, samples were diluted prior to analysis with Milli-Q water (1:10).
Nanoemulsions were observed by negative-staining electron microscopy as a direct measurement of their droplet size and shape as reported by Artiga-Artigas et al. [18 (link)]. The grids were observed in a transmission electron microscope Morgagni 268D TEM (FEI Company, Netherlands) with a CCD Mega-View camera (Olympus, Tokyo, Japan).
Cells THP-1 (human leukemia monocytic cell line) and HT-29 (human colon cancer cell line) were used to evaluate the cytotoxicity of coatings. Cell culture maintenance and cell viability were evaluated as described previously [4 (link)].
Nanoemulsions were observed by negative-staining electron microscopy as a direct measurement of their droplet size and shape as reported by Artiga-Artigas et al. [18 (link)]. The grids were observed in a transmission electron microscope Morgagni 268D TEM (FEI Company, Netherlands) with a CCD Mega-View camera (Olympus, Tokyo, Japan).
Cells THP-1 (human leukemia monocytic cell line) and HT-29 (human colon cancer cell line) were used to evaluate the cytotoxicity of coatings. Cell culture maintenance and cell viability were evaluated as described previously [4 (link)].
10
Cohesin Trimer Preparation and Imaging
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Cohesin trimer was first diluted to a concentration of approximately 0.1 mg mL−1 in 50 mM sodium phosphate buffer pH 7.6 (including 150 mM NaCl, 5% glycerol and 0.5 mM TCEP) and subsequently diluted 1:1 in spraying buffer, containing 200 mM ammonium acetate and 60% (v/v) glycerol, pH adjusted to 7.6. After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with 0.7 nm Platinum (BALTIC, Germany) at an angle of 4-5°, followed by 7 nm Carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and inspected in an FEI Morgagni 268D TEM (FEI, The Netherlands) operated at 80 kV. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
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