For Transmission electron microscopy (TEM), MEF cells were cultured on aclar foil and fixed with pre-warmed fixative solution (2% glutaraldehyde, 2.5% sucrose, 3 mM CaCl2, 100 Mm HEPES, pH 7.3) at RT for 30 min and 4°C for another 30 min. Afterward, fixed cells were washed with 0.1 M sodium cacodylate buffer, incubated with 1% OsO4, 1.25% Sucrose, 10 mg/ml Potassium ferrocyanide in 0.1 M Cacodylate buffer for 1 h on ice, and washed three times with 0.1 M Cacodylate buffer. Subsequently, cells were dehydrated using ascending ethanol series (50, 70, 90, 3 × 100) for 7 min each at 4°C. After that, cells were incubated with ascending EPON series at 4°C: EPON with ethanol (1 + 1) for 1 h, EPON with ethanol (3 + 1) for 2 h, EPON alone ON, 2 × 2 h with fresh EPON at RT and finally embedded for 48–72 h at 62°C. Ultrathin sections of 70 nm were cut with a diamond knife using an ultramicrotome (Leica, UC7) and stained with uranyl acetate for 15 min at 37°C and lead nitrate solution for 4 min. Electron micrographs were taken with a JEM-2100 Plus Electron Microscope (JEOL). Camera OneView 4K 16 bit (Gatan), and software DigitalMicrograph (Gatan).
Oneview 4k 16 bit
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OneView 4K 16 bit is a high-resolution imaging solution that captures images at 4K resolution with 16-bit depth. It provides detailed visual information for various applications.
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3 protocols using oneview 4k 16 bit
1
Transmission Electron Microscopy of MEF Cells
2
Electron Microscopy Sample Preparation
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Neurons cultured on 18 mm Ø coverslips were fixed with pre-warmed fixative solution (2% glutaraldehyde, 2.5% sucrose, 3 mM CaCl2, 100 mM HEPES, pH 7.4) at RT for 30 min, followed by the post-fixation at 4 °C for 30 min. Afterwards, the cells were washed with 0.1 M sodium cacodylate buffer, incubated with 1% OsO4, 1.25% sucrose, 10 mg/ml K4[Fe(CN)6]·3 H2O in 0.1 M cacodylate buffer for 1 h on ice and washed three times with 0.1 M cacodylate buffer. Subsequently, the cells were dehydrated at 4 °C using ascending ethanol series (50, 70, 90, 100%, 7 min each), incubated with ascending EPON series (EPON with ethanol (1 + 1) for 1 h; EPON with ethanol (3 + 1) for 2 h; EPON alone ON; 2 × 2 h with fresh EPON at RT) and finally embedded for 48–72 h at 62 °C. Coverslips were removed with liquid nitrogen and heat, consecutively. Ultrathin sections of 70 nm were made using an ultramicrotome (Leica, UC7) and stained with uranyl acetate for 15 min at 37 °C and lead nitrate solution for 4 min. Electron micrographs were taken with a JEM-2100 Plus Electron Microscope (JEOL), Camera OneView 4 K 16 bit (Gatan) and software DigitalMicrograph (Gatan).
3
Ultrastructural Analysis of Mitochondria
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Cell samples were fixed in 2.5% glutaraldehyde for 2 h at room temperature and then stored at 4 °C overnight. Subsequently, samples were post-fixed with 1% osmium tetroxide for 1 h, washed, dehydrated through an ethanol gradient (30, 50, 70 and 95%, 5 min per step), embedded and polymerized at 60 °C for 48 h. Ultrathin sections of 85 nm were cut and observed in a Tecnai 12 BioTwin transmission electron microscope (FEI Company, Eindhoven, The Netherlands) at 120 keV.
For tissue microscopy, the T10 segment of spinal cord was isolated and sliced in 1 mm thick longitudinal sections. The fixed tissues were embedded using EPON as previously descried. Ultrathin sections of 70 nm were cut using an ultramicrotome (Leica Microsystems, UC6) with a diamond knife (Diatome, Biel, Switzerland) and stained with 1.5% uranyl acetate at 37 °C for 15 min and lead citrate solution for 4 min. Electron micrographs were taken with a JEM-2100 Plus Transmission Electron Microscope (JEOL), equipped with Camera OneView 4 K 16 bit (Gatan) and software Digital Micrograph (Gatan).
For analysis, the length and morphology of each mitochondrion was determined in ImageJ following manual drawing of single organelle. All parameters obtained from one field of view were averaged together.
For tissue microscopy, the T10 segment of spinal cord was isolated and sliced in 1 mm thick longitudinal sections. The fixed tissues were embedded using EPON as previously descried. Ultrathin sections of 70 nm were cut using an ultramicrotome (Leica Microsystems, UC6) with a diamond knife (Diatome, Biel, Switzerland) and stained with 1.5% uranyl acetate at 37 °C for 15 min and lead citrate solution for 4 min. Electron micrographs were taken with a JEM-2100 Plus Transmission Electron Microscope (JEOL), equipped with Camera OneView 4 K 16 bit (Gatan) and software Digital Micrograph (Gatan).
For analysis, the length and morphology of each mitochondrion was determined in ImageJ following manual drawing of single organelle. All parameters obtained from one field of view were averaged together.
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