Heart tissues were fixed with 2.5% glutaraldehyde, 2.5% polyvidone 25 and 0.1 M sodium cacodylate (pH 7.4). After being washed with 0.1 M sodium cacodylate buffer (pH 7.4), the samples were post-fixed in the same buffer containing 2% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h [8 ]. The samples were then washed in water, contrasted en bloc with uranyl acetate, dehydrated using an ascending ethanol series and embedded in a Durcupan ACM-based resin [29 ]. Ultrathin sections were cut with a Reichert Ultracut S ultramicrotome (Science Service, Munich, Germany) and contrasted with lead citrate. Images were viewed and captured with an EM 10 CR electron microscope (Carl Zeiss Gemini, Germany), and were analyzed by an independent blinded investigator [30 (link)].
Em 10 cr electron microscope
Manufactured by Zeiss
Sourced in Germany
The EM 10 CR is an electron microscope designed for high-resolution imaging of samples. It utilizes a focused beam of electrons to magnify and image the fine details of specimens. The core function of the EM 10 CR is to provide users with a powerful tool for detailed microscopic analysis and observation.
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6 protocols using em 10 cr electron microscope
1
Transmission Electron Microscopy of Heart Tissue
2
Microscopic Techniques for Tissue Analysis
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Light and fluorescence microscopies were performed using epifluorescent microscopes fitted with differential interference contrast (DIC) optics (Imager Z1, Zeiss) fitted with a black and white camera (Axiocam MRm, Zeiss). Images were pseudo-colorized digitally (Adobe Photoshop).
For transmission electron microscopy (TEM), blocks were prepared from 10 ovaries or 10 venom glands per sample. Dissected samples were pooled into 100 μl of Ringer’s saline (KCl 182 mM; NaCl 46 mM; CaCl2 3 mM; Tris-HCl 10 mM) in a centrifuge vial on ice. An equivalent volume of fixative (4% glutaraldehyde (Sigma) in 0.2 M sodium cacodylate buffer, pH 7.2) was then added and the sample was kept for 24 h at 4 °C. Fixed samples were centrifuged (500 × g, 10 min) to pellet tissues and remove the fixative prior to post-fixation in 2% osmium tetroxide in cacodylate buffer. Following dehydration in graded series of ethanol solutions, samples were embedded in Epon. Sample sections were cut with a diamond knife using a LKB ultramicrotome, mounted on copper grids, stained with uranyl acetate and lead citrate, and observed with a Zeiss EM10CR electron microscope at 80 kV.
For transmission electron microscopy (TEM), blocks were prepared from 10 ovaries or 10 venom glands per sample. Dissected samples were pooled into 100 μl of Ringer’s saline (KCl 182 mM; NaCl 46 mM; CaCl2 3 mM; Tris-HCl 10 mM) in a centrifuge vial on ice. An equivalent volume of fixative (4% glutaraldehyde (Sigma) in 0.2 M sodium cacodylate buffer, pH 7.2) was then added and the sample was kept for 24 h at 4 °C. Fixed samples were centrifuged (500 × g, 10 min) to pellet tissues and remove the fixative prior to post-fixation in 2% osmium tetroxide in cacodylate buffer. Following dehydration in graded series of ethanol solutions, samples were embedded in Epon. Sample sections were cut with a diamond knife using a LKB ultramicrotome, mounted on copper grids, stained with uranyl acetate and lead citrate, and observed with a Zeiss EM10CR electron microscope at 80 kV.
3
Ultrastructural Analysis of COLO205 and HCT116 Cells
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COLO205 and HCT116 cells were collected, suspended and fixed overnight at 4°C in 2% glutaraldehyde (http://www.tianld.com/djhc ) and 1% tannic acid (403,040, Sigma) prepared in 0.1 M sodium cacodylate (pH 7.3, C0250, Sigma). The cells were washed three times in sodium cacodylate buffer and incubated in 2% osmium tetroxide (http://www.tianld.com/djhc ) for 2 h at room temperature. Next, the cells were washed thrice in ddH2O and exposed to 1% uranyl acetate (http://www.tianld.com/djhc ) in water for 15 min at room temperature. Subsequently, the samples were dehydrated using an ascending ethanol series and embedded in Spurr's low‐viscosity medium (http://www.tianld.com/djhc ). Ultrathin sections were cut using a Reichert Ultracut S ultramicrotome (Science Service, Munich, Germany) and contrasted with lead citrate. Images were viewed and captured using an EM 10 CR electron microscope (Carl Zeiss Gemini, Germany).
4
Electron Microscopy of Extracellular Vesicles
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Isolated and pelleted EVs were fixed in glutaraldehyde-PBS (2% final). After washing and post-fixing in osmium tetroxide (2%) and K4[Fe(CN)6] (1.5%), samples were totally enclosed in contrast solution uranyl acetate, dehydrated with a graded dilution series of ethanol, and finally embedded into glycid-ether-100-based resin. Ultrathin sections were cut (Reichert Ultracut S ultramicrotome, Leica Microsystems). Slices were contrasted and analyzed with a Zeiss EM 10 CR electron microscope.
5
Quantitative Ultrastructural Analysis of Renal Glomeruli
6
Ultrastructural Analysis of Renal Cortex
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Transmission electron microscopy (TEM) was performed at the Institute for Clinical Chemistry and Pathobiochemistry, Otto-von-Guericke-University Magdeburg and at the Institute of Anatomy, University Leipzig, Germany. Renal cortex tissues were fixed with 2.5% glutaraldehyde, 2.5% polyvidone 25, 0.1 M sodium cacodylate pH 7.4. After washing with 0.1 M sodium cacodylate buffer (pH 7.4), samples were post-fixed in the same buffer containing 2% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h, washed in water, contrasted en bloc with uranyl acetate, dehydrated using an ascending series of ethanol and embedded in glycidyl ether 100-based resin. Ultrathin sections were cut with a Reichert Ultracut S ultramicrotome (Leica Microsystems, Wetzlar, Germany), contrasted with uranyl acetate and lead citrate and were viewed with an EM 10 CR electron microscope (Carl Zeiss NTS, Oberkochen, Germany). The GBM thickness was analysed by NIH-ImageJ software56 (link).
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