For TEM, a TiO2 NPs suspension (500 µg/mL; 5 µL) was applied onto glow-discharge-activated [22 (link)] carbon-coated 400-mesh copper grids (G400, SPI Supplies, Structure Probe, Inc., West Chester, PA, USA). Nanoparticles were sedimented for 1 min and the remaining solution was then blotted with filter paper and the grids were air-dried. A Philips CM100 electron microscope (Philips EO, Eindhoven, The Netherlands; Thermo Fisher Scientific) equipped with a Veleta slow-scan CCD camera (EMSIS GmbH, Muenster, Germany) was used to examine the grids. TEM images were processed in the proprietary iTEM software (EMSIS GmbH, Muenster, Germany).
Item software
Manufactured by EMSIS
Sourced in Germany
ITEM software is a comprehensive data management solution designed for laboratory environments. It provides a centralized platform for organizing and analyzing laboratory data, enabling efficient data storage, retrieval, and reporting.
Automatically generated - may contain errors
Lab products found in correlation
Poly bed 812 resin, by Polysciences (8 mentions)
Em910 electron microscope, by Zeiss (7 mentions)
Quemesa cdd camera, by EMSIS (6 mentions)
Quemesa ccd camera, by EMSIS (4 mentions)
Morada ccd camera, by EMSIS (3 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (2 mentions)
Epon 812, by TAAB (2 mentions)
Jem 1400, by Olympus (2 mentions)
Poly bedr 812, by Polysciences (2 mentions)
Morgagni electron microscope, by EMSIS (2 mentions)
Quemesa, by EMSIS (2 mentions)
Reichert ultracut s ultramicrotome, by Leica (2 mentions)
Tecnai spirit electron microscope, by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Veleta slow scan ccd camera, by EMSIS (1 mentions)
Cm100 electron microscope, by Philips (1 mentions)
Ultracut uct microtome, by Leica (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Reichert ultracut s, by Leica (1 mentions)
30 protocols using item software
1
Characterization of TiO2 Nanoparticles by TEM
2
Ultrastructural Analysis of Tumor Tissues
3
Ultrastructural Analysis of Photosynthetic Cells
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Cells incubated in darkness, normal or high light for 6 hours, as well as gametic cells and zygotes from the crosses were concentrated, deposited on polylysine-coated cover slips, fixed with a mixture of 2% (wt/vol) paraformaldehyde, 1% (wt/vol) glutaraldehyde in 0.2 M phosphate buffer (PB), pH 7.4, post-fixed with 1% (wt/vol) OsO4 supplemented with 1.5% (wt/vol) potassium ferrocyanide, dehydrated in ethanol, and embedded in epon 812 (TAAB Laboratories Equipment). Ultrathin sections were prepared with a Reichert UltracutS ultramicrotome (Leica), counter-stained with uranyl acetate, and viewed at 80kV with a Transmission Electron Microscope (TEM; Tecnai Spirit G2; Thermo Fisher Scientific, Eindhoven, The Netherlands) equipped with a QUEMESA CCD 4K camera (EMSIS GmbH, Münster, Germany) using iTEM software (EMSIS).
4
Ultrastructural Analysis of Cells
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Culture media was removed and cells were washed once with PBS, followed by fixation with 2.5% glutaraldehyde (Merck) in 0.1 M cacodylate buffer, pH7.4, overnight at 4 °C. Samples were washed three times with 0.1 M cacodylate buffer and post-fixed by 1% OsO4/1% Ru(CN)6 for 90 min at RT. After washing with 0.1 M cacodylate buffer, a series of increasing ethanol concentrations (30%-100%) were used to dehydrate the samples. Samples were embedded in EPON and polymerized for 48 h at 65 °C. The EPON-embedded samples were removed from the substrate by breaking the 96-well plate in a vise. Regions with highest cell density were determined by light microscopy and ultrathin sections of (80 nm) were cut using an Ultracut UCT microtome (Leica). Sections were collected on single-slot, formvar-coated copper grids and counterstained with 0.5% uranyl acetate for 25 min and lead cytrate for 3 min at RT in an automated section stainer (Leica). Images were captured on a JEOL 1010 transmission EM at 60 kV with a side-mounted CCD camera (Modera, EMSIS) controlled by the iTEM software (EMSIS GmbH, Germany) at 40 K or 80 K magnification. EM data are supported by 2 independent experiments.
5
Ultrastructural Analysis of Yeast Cells
6
Ultrastructural Analysis of Hepatocyte Mitochondria
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Freshly isolated tissue samples were punched with 1 mm³ for 3–5 times and immediately fixed by immersion with 2% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer at RT for 2 h and at 4°C overnight. Samples were transferred to 1% paraformaldehyde in 0.1 M phosphate buffer at 4°C for long-term storage and then postfixed and stained consecutively with 1% (w/v) tannic acid, 1% (v/v) osmium tetroxide, and 2% (w/v) uranylacetate at RT, dehydrated in a graded series of ethanol, and embedded in PolyBed® 812 resin (Polysciences). Ultrathin sections (60–80 nm) were made and stained with uranyl acetate and lead citrate, and examined at 80 kV with a Zeiss EM 910 electron microscope (Zeiss). Acquisition was done with a Quemesa CCD camera using iTEM software (Emsis GmbH) at 5000× magnification with a field of view of 11.2 × 7.4 μm per image. 9 independent images per mouse at different positions within the liver sections representing hepatocytes were used for evaluation and quantification of mitochondria.
7
Quantitative Analysis of Vascular Caveolae
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Quantitative assessment of caveolae was carried out as previously described (Lowalekar et al., 2012 ). Isolated VSMCs from mesenteric arteries were dehydrated in a graded series of ethanol and embedded in the PolyBed® 812 resin (Polysciences Europe GmbH), ultrathin sections (60–80 nm) were cut (Leica microsystems), and uranyl acetate and lead citrate staining was performed. Samples were examined at 80 kV with a Zeiss EM 910 electron microscope (Zeiss), and image acquisition was performed with a Quemesa CDD camera and the iTEM software (Emsis GmbH). The density of caveolae was calculated as number of caveolae per µm. The diameter of caveolae neck (nm) and caveolae size (nm) were determined by using the parallel dimension function of CorelDRAW. Values from all electron microscopy images (n = 18 cells in each group) were averaged for each group.
8
Transmission Electron Microscopy of Phage
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The phage particles were concentrated by centrifugation at 16,000× g for 90 min at 4 °C using an Eppendorf centrifuge (5415R, rotor model 3328, Enfield, NJ, USA). A carbon-coated copper grid was incubated with a drop of the phage preparation for one minute and then stained with 1% uranyl acetate (pH about 4.2) for 30 s. The micrographs were then captured using a transmission electron microscope (JEOL JEM-1400, Tokyo, Japan, 80 kV) with an Olympus Morada CCD-camera (Olympus, Tokyo, Japan), operating at iTEM software (EMSIS GmbH, Muenster, Germany).
9
Ultrastructural Analysis of MNT-1 Cells
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MNT-1 cells cultured on carbonated sapphire disks or CryoCapsules (CryoCapCell; Heiligenstein et al., 2014 (link)) were transfected with control, Myo6, or WASH siRNAs or treated for 60 min with 25 µM DMSO or TIP. Cell were high-pressure frozen with HPM100 (Leica Microsystems) or HPM Live μ (CryoCapCell) and then freeze substituted in anhydrous acetone containing 1% OsO4/2% H2O for 64 h in a freeze substitution system (AFS; Leica Microsystems). Cells were embedded in epon 812 (TAAB Laboratories Equipment) and processed for sectioning and contrasting with uranyl acetate and lead citrate. Ultrathin sections were observed at 80 kV transmission electron microscope (Tecnai Spirit G2; Thermo Fisher Scientific) equipped with a QUEMESA CCD camera (EMSIS) using iTEM software (EMSIS). For ultrathin cryosections and immunogold labeling, MNT-1 cells were grown on six-well plates and fixed with 2% PFA/0.2% glutaraldehyde/0.1 M phosphate buffer. Cells pellets were embedded in 10% gelatin and infused in 2.3 M sucrose. Gelatin blocks were frozen and processed for ultracryomicrotomy. Ultrathin sections (90 nm) were double-immunogold labeled using PAG 10 or 15 nm and analyzed by EM as described above.
10
Histology and TEM Fixation Protocol
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For histology and TEM, heads of 5 dpf larvae were fixed in 2% glutaraldehyde, 2% paraformaldehyde in 50 mM Hepes (pH 7.25) for 1.5 h at room temperature (RT), then at 4°C overnight. Heads were washed 5×3 min in Hepes buffer, 2×5 min in PBS, incubated for 60 min in 1% OsO4/1.5% potassium ferrocyanide in PBS, washed 2×5 min in distilled water and dehydrated in an ethanol series. Samples were incubated 2×10 min in propidium oxide and embedded in Durcupan. For histology, semithin sections of 500 nm were stained with 1% Toluidine Blue, 0.5% sodium borate solution and mounted in Entellan (Merck). Samples were imaged with Zeiss Imager.Z1 microscope using AxioCam HRc and AxioVision software (Zeiss). For TEM, ultrathin sections of 70 nm were stained with uranyl acetate and lead citrate, and imaged with a Morgagni TEM (80 kV) using a Morada CCD camera (EMSIS GmbH) and ITEM software (EMSIS GmbH). Images were processed using TrakEM2 in Fiji (Cardona et al., 2012 (link); Schindelin et al., 2012 (link)) and Adobe Photoshop.
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