Gonads samples for TEM examination were fixed in 2.5% glutaraldehyde (Serva Electrophoresis, Heidelberg, Germany). After 24 hours, the specimens were washed in the cacodylate or phosphate buffers (0.1 M, pH 7.4, Serva) and postfixed for 1 h in 1% osmium tetroxide (Serva). Subsequently, the specimens were dehydrated in ethyl alcohol and twice in pure acetone (Chempur, Poland). Afterwards samples were embedded in epoxy resin (Epon 812, Serva). Epon blocks were cut into semithin (600 nm thick) sections and stained with toluidine blue (Serva). Finally, ultrathin (50 nm thick) sections were prepared using an ultramicrotomes Power Tome XL (RMC, Tucson, USA) and Reichert Ultracut E. Ultrathin sections were counterstained with uranyl acetate and lead citrate (Serva), and examined in a TEM JEM-1011 (JEOL,Tokyo, Japan) or Zeiss EM 900. Digital micrographs were collected with the use of TEM imaging platform iTEM1233 equipped with the Morada Camera (Olympus, Münster, Germany).
Item 1233
Manufactured by Olympus
Sourced in Germany, Japan
ITEM 1233 software is a data analysis tool designed for use with Olympus lab equipment. The software provides basic functionality for processing and visualizing data collected from Olympus instruments.
Automatically generated - may contain errors
Lab products found in correlation
Morada camera, by Olympus (3 mentions)
Jem 1200ex, by JEOL (3 mentions)
Jem 1011 tem, by JEOL (2 mentions)
Glutaraldehyde, by Serva Electrophoresis (2 mentions)
Toluidine blue, by Serva Electrophoresis (2 mentions)
Osmium tetroxide, by Serva Electrophoresis (2 mentions)
Epon 812, by Serva Electrophoresis (2 mentions)
Uranyl acetate and lead citrate, by Serva Electrophoresis (1 mentions)
Cacodylate buffer, by Serva Electrophoresis (1 mentions)
Uranyl acetate, by Serva Electrophoresis (1 mentions)
Lead citrate, by Serva Electrophoresis (1 mentions)
Morada, by Olympus (1 mentions)
Silver citrate, by Merck Group (1 mentions)
Agnps, by JEOL (1 mentions)
6 protocols using item 1233
1
Ultrastructural Analysis of Gonad Tissues
2
Ultrastructural Analysis of Skeletal Muscle
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For TEM examination skeletal muscle samples from six patients were fixed in 2.5% glutaraldehyde (Serva Electrophoresis, Heidelberg, Germany). After 24 h, the specimens were washed in cacodylate buffer (0.1 M, pH 7.4, Serva) and postfixed for 1 h in 1% osmium tetroxide (Serva). Subsequently, they were rinsed with the cacodylate buffer. Afterwards, the specimens were dehydrated in highly concentrated ethyl alcohol and twice in pure acetone (Chempur, Piekary Slaskie, Poland). Samples were embedded in epoxy resin (Epon 812, Serva). To select the examined area for TEM Epon blocks were cut into semithin, 600-nm-thick sections, stained with toluidine blue (Serva) and closed with the use of Euparal mounting agent (Roth, Mannheim, Germany). Finally, ultrathin, 50-nm-thick sections were prepared using an ultramicrotome Power Tome XL (RMC, Tucson, USA).
To improve the contrast, ultrathin sections placed on the copper grids were counterstained with uranyl acetate and lead citrate (Serva), and examined in the TEM JEM-1011 (JEOL, Tokyo, Japan). Digital micrographs were prepared with the use of TEM imaging platform iTEM1233 equipped with a Morada Camera (Olympus, Münster, Germany) at magnifications ranging from 5 to 20 K.
To improve the contrast, ultrathin sections placed on the copper grids were counterstained with uranyl acetate and lead citrate (Serva), and examined in the TEM JEM-1011 (JEOL, Tokyo, Japan). Digital micrographs were prepared with the use of TEM imaging platform iTEM1233 equipped with a Morada Camera (Olympus, Münster, Germany) at magnifications ranging from 5 to 20 K.
3
Characterization of Silver Nanoparticles
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AgNPs (CAS No. 730785) and silver citrate (a source of silver ions) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The form of these AgNPs is defined by the manufacturer as a colloidal solution of nanoparticles (10 ± 4 nm in diameter) suspended in an aqueous citrate buffer in a concentration of 0.02 mg AgNPs/mL to provide long-term stability. According to the manufacturer, each batch of AgNPs is characterized to ensure a homogeneous product (monodisperse AgNPs free from agglomeration), as indicated by the following parameters: refractive index n20/D = 1.333, fluorescence—λem = 388 nm, and full width at half maximum value (FWHM) = 59 nm.
Additional characterization of these AgNPs was published in our previous study where we assessed the degree of dispersion and size distribution of AgNPs with a transmission electron microscope (JEM-1200EX, Jeol, Japan) equipped with a digital camera MORADA and iTEM 1233 software (Olympus Soft Imaging Solutions, GmbH, Germany) (Skalska et al. 2015 ).
Additional characterization of these AgNPs was published in our previous study where we assessed the degree of dispersion and size distribution of AgNPs with a transmission electron microscope (JEM-1200EX, Jeol, Japan) equipped with a digital camera MORADA and iTEM 1233 software (Olympus Soft Imaging Solutions, GmbH, Germany) (Skalska et al. 2015 ).
4
Ultrastructural Analysis of Rat Brain
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After deep anesthesia with nembutal (80 mg/kg b.w.), the rats were perfused through the ascending aorta with 0.9% NaCl in 0.01 M sodium-potassium phosphate buffer (pH 7.4) and then with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Brain samples were collected, fixed in the above ice-cold fixative solution, and post-fixed in 1% OsO4 solution. Then the samples were dehydrated in the ethanol gradient, embedded in epoxy resin (Epon 812) and cut into ultrathin sections, which were stained with 9% uranyl acetate and lead nitrate. Analysis of brain sections taken from cerebral cortex and hippocampus was performed by TEM (JEM-1200EX, Jeol, Japan) equipped with a digital camera MORADA and iTEM 1233 software (Olympus Soft Imaging Solutions, GmbH, Germany).
Additionally, another group of sections was not contrasting with metallic compounds (Pb, U) to avoid occurrence of osmium artifacts or lead precipitates. Omitting of this step allows visualization of the potential presence of silver in brain tissue, which is the only metal found in cellular structures during such tissue processing.
Additionally, another group of sections was not contrasting with metallic compounds (Pb, U) to avoid occurrence of osmium artifacts or lead precipitates. Omitting of this step allows visualization of the potential presence of silver in brain tissue, which is the only metal found in cellular structures during such tissue processing.
5
Ultrastructural Lung Tissue Analysis
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All reagents were purchased from Sigma-Aldrich. After sacrifice of mice by isoflurane overdose, the 2% paraformaldehyde was instilled overnight at room temperature; and after ligation of trachea, the lungs were excised. The samples were sequentially fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in cacodylate buffer, and 1% osmium tetraoxide with potassium ferricyanide at room temperature for 2 hours. The lung samples were postfixed in a 1% OsO 4 solution for 2 hours, then dehydrated in a series of ethanol solutions and embedded in resin. Ultrathin sections were stained with uranyl acetate and lead citrate. The images were acquired using a JEM-1011 EX transmission electron microscope (Jeol, Tokyo, Japan) equipped with a MOR-ADA camera and iTEM 1233 software (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
6
Electron Microscopic Analysis of Rat Brain
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The rats were anesthetized with nembutal (80 mg/kg b.w.) and perfused first with 0.9% NaCl in 0.01 M sodium–potassium phosphate buffer (pH 7.4), and then with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Brain samples were subjected to a routine method of tissue processing for electron microscopic analysis. The samples were post-fixed in 1% OsO4 solution, dehydrated in an ethanol gradient, embedded in epoxy resin (Epon 812), and cut into ultra-thin sections stained with 9% uranyl acetate and lead nitrate. A transmission electron microscope (TEM) (JEM-1200EX, Jeol, Tokyo, Japan) equipped with a digital camera MORADA and iTEM 1233 software (Olympus Soft Imaging Solutions, GmbH, Münster, Germany) was used for the study.
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