Cells were fixed in 3% glutaraldehyde (Agar scientific, Wetzlar, Germany) for at least 4 h, washed in 0.1 M Soerensen’s phosphate buffer (Merck, Darmstadt, Germany), and embedded into 2.5% agarose (Sigma, Steinheim, Germany). agarose cubes were rinsed in 0.1 M Soerensen’s phosphate buffer. Post-fixation of cells was performed in 1% OsO4 in 17% sucrose buffer. After fixation, cells were washed in 17% sucrose buffer and deionized water and dehydrated by an ethanol series (30, 50, 70, 90 and 100%) for 10 min each and the last step thrice. The dehydrated specimens were incubated in propylene oxide (Serva, Heidelberg, Germany) for 30 min, in a mixture of Epon resin (Sigma) and propylene oxide (1:1) for 1 h and finally in pure Epon for 1 h. Samples were embedded in pure Epon. Epon polymerization was performed at 37°C for 12 h and then 80°C for 48 h. Ultrathin sections (70-100nm) were cut with a diamond knife (Leica, Wetzlar, Germany) by an ultramicrotome (Reichert Ultradcut S, Leica) and picked up on Cu/Rh grids (HR23 Maxtaform, Plano, Wetzlar, Germany). Negative staining by uranyl acetate and lead citrate (all EMS, Munich, Germany) was performed to enhance TEM contrast. The specimens were viewed using a Zeiss Leo-906 electron microscope (Oberkochen, Germany), operated at an acceleration voltage of 60kV.
Leo 906 electron microscope
Manufactured by Zeiss
Sourced in Germany
The LEO 906 is an electron microscope designed and manufactured by Zeiss. It uses a beam of electrons to magnify and image the surface of samples. The LEO 906 allows users to observe the detailed structure and composition of materials at high resolutions.
Automatically generated - may contain errors
Lab products found in correlation
Diamond knife, by Leica (1 mentions)
Propylene oxide, by Merck Group (1 mentions)
Glutaraldehyde, by Agar Scientific (1 mentions)
Agarose, by Merck Group (1 mentions)
Propylene oxide, by Serva Electrophoresis (1 mentions)
Soerensen s phosphate buffer, by Merck Group (1 mentions)
Epon resin, by Merck Group (1 mentions)
Pioloform, by Ted Pella (1 mentions)
Particle size analyzer, by Malvern Panalytical (1 mentions)
Ftir prestige 21, by Shimadzu (1 mentions)
Epon 812 resin, by TAAB (1 mentions)
Araldite resin, by Electron Microscopy Sciences (1 mentions)
Axioskop2 mot plus microscope, by Zeiss (1 mentions)
Aqueous uranyl acetate, by Serva Electrophoresis (1 mentions)
Alcian blue, by Merck Group (1 mentions)
8 protocols using leo 906 electron microscope
1
Electron Microscopy Sample Preparation
2
Ultrastructural Analysis of HEK293 Cells
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HEK293 and HEK293/SET cells were collected after trypsin digestion, washed in fresh PBS (pH 7.4), fixed by immersion in glutaraldehyde, post-fixed in osmium tetroxide, stained in block with uranium acetate, dehydrated, and embedded in resin (Embed 812, EM Sciences). Ultrathin sections (60 nm) were collected on Pioloform (Ted Pella, Redding, CA) and carbon-coated single sloth grids and contrasted with uranyl acetate and lead citrate. Cells were examined on a ZEISS LEO 906 electron microscope.
3
Characterization of Nanomaterial Properties
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Fluorescence spectra were recorded by a Hitachi (Tokyo, Japan). The morphology and particle size of the nanomaterials were determined by field emission scanning electron microscopy (FESEM) through a FEG-SEM MIRA3 TESCAN (Brno, Czech Republic) and transmission electron microscopy (TEM) by Carl Zeiss LEO 906 electron microscope operated at 100 kV (Oberkochen, Germany), respectively. Energy-dispersive X-ray (EDX) analysis was also determined by the FESEM technique. Fourier-transform infrared spectroscopy (FTIR) spectra were captured through a Shimadzu model FTIR prestige 21 (Tokyo, Japan). Surface charge and nanoparticle sizes were determined by a Malvern particle size analyzer (Malvern, UK). The MTT assay was measured at 570 nm by an enzyme-linked immunosorbent assay (ELISA) plate-reader (Florida, USA). Fluorescence microscopy images were captured by an Olympus microscope Model Bh2-RFCA (Tokyo, Japan).
4
Ultrastructural Analysis of Apoptosis in Rat Hippocampus
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The deeply anesthetized rats were transcardially per-fused with 200 mL of normal saline followed by 200 mL of paraformaldehyde 4% in PBS (0.1 M, pH 7.4) at 4°C. The hippocampi were removed and immersed in glutaraldehyde 2.5%. The specimens were then washed in PBS and postfixed in OsO4 1% for 2 h at room temperature.
After dehydration in ascending ethanols, they were embedded in Epon 812 resin (TAAB, UK) and polymerized for 48 h at 55°C. Semi-thin sections (0.3 μm) were stained with toluidine blue to identify CA1 pyramidal layer. Subsequently, 70 nm sections were cut and stained with uranyl acetate and lead. The sections were examined with a LEO 906 electron microscope (Zeiss, Germany). Ultrathin sections were analyzed to find changes in neuronal ultrastructure consistent with apoptosis, including swelling and vacuolization of mitochondria, nuclear pyknosis, and margination of nucleoli (Kermanian et al., 2012 ).
After dehydration in ascending ethanols, they were embedded in Epon 812 resin (TAAB, UK) and polymerized for 48 h at 55°C. Semi-thin sections (0.3 μm) were stained with toluidine blue to identify CA1 pyramidal layer. Subsequently, 70 nm sections were cut and stained with uranyl acetate and lead. The sections were examined with a LEO 906 electron microscope (Zeiss, Germany). Ultrathin sections were analyzed to find changes in neuronal ultrastructure consistent with apoptosis, including swelling and vacuolization of mitochondria, nuclear pyknosis, and margination of nucleoli (Kermanian et al., 2012 ).
5
Ultrastructural Analysis of SK-MEL-37 Cells
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SK-MEL-37 cells were starved with EBSS and later fixed in a 2% paraformaldehyde, 2% glutaraldehyde solution (1:1) in sodium cacodylate buffer 0.1 M (pH 7.4) for 24 h at 4°C. The cells were subsequently post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series and embedded in Araldite resin (Electron Microscopy Science). Ultrathin sections (~90 nm) were stained with uranyl acetate and lead citrate and examined using a ZEISS Leo 906 electron microscope (Carl Zeiss).
6
Localization of Endogenous Gal-1 Protein in ARPE Cells
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To detect the localization of the endogenous Gal-1 protein, ultrathin sections (70 nm) of ARPE cells were sequentially incubated with the following reagents at room temperature: i) distilled water; ii) PBS containing 1% egg albumin (1% PBEA) for 10 min; iii) 5% PBEA; iv) rabbit polyclonal anti-Gal-1 Ab (1:100; Zymed Laboratories) for 2 h, with normal rabbit serum as a control (1:100); and v) 1% PBEA containing 0.01% Tween-20 in three washes (5 min each). To detect Gal-1, a goat anti-rabbit IgG Ab (1:50 in 1% PBEA) conjugated with 15 nm colloidal gold (British Biocell, Cardiff, UK) was applied. After 1 h, the sections were washed thoroughly in 1% PBEA and then in distilled water. These sections were stained with uranyl acetate and lead citrate and then examined using a ZEISS Leo 906 electron microscope (Carl Zeiss). Randomly photographed sections of ARPE cells were used for immunocytochemical analysis. The area of the cell compartment was determined with AxioVision software on an Axioskop 2-Mot Plus Zeiss microscope. The density of immunogold particles (number of gold particles per μm2) was calculated and expressed for each cell compartment. Values are reported as the mean ± SEM of 10 electron micrographs analyzed per group.
7
Electron Microscopy Visualization of Extracellular Vesicles
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EVs were fixed in a 2% paraformaldehyde, 2% glutaraldehyde solution (1:1) in sodium cacodylate buffer (pH 7.4) for 30 min at 4 °C, followed by washing with PBS and ultracentrifugation at 100,000× g. Next, EVs (10 µL) were mounted in TEM grids, contrasted with uranyl acetate 2% for 15 min, and washed in distilled water. Fluid excess was blotted in filter paper, air-dried, and examined using a ZEISS Leo 906 electron microscope (Carl Zeiss, Jena, Germany).
8
Negative Staining Electron Microscopy
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For the negative staining, carbon-coated mesh grids were hydrophilized with Alcian Blue (Sigma-Aldrich, St. Louis) solution (1% in 1% acetic acid in water) for 10 min, followed by washing steps on drops of distilled H2O. The grids were placed on 15 μL of particle solution for 10 min, followed by removing the solution by a filter paper, washing steps on drops of distilled H2O and finally placed on a drop of freshly prepared solution of 1% aqueous uranyl acetate (Serva, Heidelberg, Germany) for 20 s. Finally, the drop on the grid was sucked off with a filter paper and the grid dried in a grid box. The images were acquired on a Zeiss Leo 906 electron microscope (Carl Zeiss, Oberkochen, Germany) at 80 kV acceleration voltage equipped with a slow scan 2K CCD camera (TRS, Moorenweis, Germany).
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