Samples were fixed for 2 hours in 0.1 M cacodylate buffer pH 7.2, containing 2% glutaraldehyde. Specimens were then washed in the same buffer and post-fixed for 2 hours with 1% osmic acid in cacodylate buffer. After standard serial ethanol dehydration, specimens were embedded in an Epon-Araldite 812 mixture. Sections were obtained with a Reichert Ultracut S ultratome (Leica, Austria). Semi-thin sections were stained by conventional methods (crystal violet and basic fuchsin) and observed under a light microscope (Olympus, Japan) to score cells for different morphological changes. Thin sections were stained by uranyl acetate and lead citrate and observed with a Jeol 1010 EX electron microscope (Jeol, Japan).
1010 ex electron microscope
Manufactured by JEOL
Sourced in Japan
The JEOL 1010 EX is a transmission electron microscope (TEM) designed for high-resolution imaging and analysis of a wide range of materials. It features a LaB6 electron source and provides accelerating voltages up to 100 kV. The microscope is equipped with a standard CCD camera for digital image capture and can be configured with various optional detectors and accessories to enable advanced analytical capabilities.
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5 protocols using 1010 ex electron microscope
1
Ultrastructural Analysis of Cell Morphology
2
Electron Microscopy of Prefrontal Cortex
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Electron microscopy was carried out by subdissecting some of the immunostained sections of the prefrontal cortex into smaller portions. These were post-fixed with 1% osmium tetroxide, dehydrated in an ascending series of ethanol and acetone, and embedded in Araldite. Thin sections were obtained from the first 5 µm of the sections, mounted on copper grids coated with Formvar, and stained with lead citrate. They were viewed using a JEOL 1010 EX electron microscope (JEOL, Tokyo, Japan).
3
Immunostained Striatum Analysis
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Immunostained sections of the striatum were subdissected into smaller portions and stained with 1% osmium tetroxide for 1 h. They were dehydrated in an ascending series of 25, 50, 75, and 100% ethanol and acetone, and embedded in Araldite. Thin sections were obtained from the first 5 μm of the immunostained sections, mounted on Formvar-coated copper grids, and stained with lead citrate. They were viewed using a JEOL 1010EX electron microscope (JEOL, Japan).
4
Morphological Analysis of EV Suspensions
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For morphological analysis, EVs suspensions (10 μL) were fixed in 50 μL of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.6 for 1 h. Five microliters of the mix were transferred onto copper 300 Mesh Formvar-carbon-coated electron microscopy grids (Pacific Grid Tech, www.grid-tech.com ). After 20 min, grids were transferred on drops of 50 μL of 0.1 M sodium cacodylate pH 7.6 with the sample membrane side facing down. After three washing in 0.1 M sodium cacodylate, grids were negatively stained with 2% uracyl acetate in water for 10 min and air dried for 5 min. All samples were observed with a Jeol 1010 EX electron microscope (Jeol, Tokyo). Data were recorded with a MORADA digital camera system (Olympus, Tokyo).
5
Ultrastructural Analysis of Cells
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Cells encapsulated in PTFE were collected for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). TEM analysis samples were fixed for 2 hours in 0.1 M cacodylate buffer pH 7.2, containing 2% glutaraldehyde. Specimens were then washed in the same buffer and post-fixed for 2 hours with 1% osmic acid in cacodylate buffer. After standard serial ethanol dehydration, specimens were embedded in an Epon-Araldite 812 mixture. Sections were obtained with a Reichert Ultracut S ultratome (Leica). Semi-thin sections were stained by conventional methods (crystal violet and basic fuchsin) and observed under a light microscope (Olympus). Thin sections were stained by uranyl acetate and lead citrate and observed with a Jeol 1010 EX electron microscope (Jeol).
For SEM, cells were fixed and dehydrated as described above, then treated with hexamethildisilazane and mounted on polylysinated slides, air dried and subsequently covered with a 9 nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech). Specimens were examined with a SEM-FEG Philips XL-30 microscope (Philips).
For SEM, cells were fixed and dehydrated as described above, then treated with hexamethildisilazane and mounted on polylysinated slides, air dried and subsequently covered with a 9 nm gold film by flash evaporation of carbon in an Emitech K 250 sputter coater (Emitech). Specimens were examined with a SEM-FEG Philips XL-30 microscope (Philips).
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