eoe sub articles

EoE Sub Articles

1. Sectioning of the Exosomes
<ol>
	<li>Prepare exosome block by embedding the purified exosome sample (obtained from the culture supernatant of HCT116 cells) in pure low viscosity embedding mixture.</li>
	<li>Section the exosome block with 60 nm thickness through an ultra-microtome.</li>
	<li>Double stain with 2% uranyl acetate for 20 min and lead citrate for 10 min. 
	For Reynolds lead solution, add 1.33 g of lead nitrate and 1.76 g of sodium citrate to a total of 50 mL distilled water.</li>
	<li>Observe the grid under transmission electron microscopy at 80 kV.</li>
	<li>Click &#34;Acquire&#34; and then click &#34;File&#34; and &#34;Save as&#34; in the CCD camera system under the electron microscope at 80 kV. Follow automatic settings for the exposure time.</li>
</ol>

<table><tbody><tr><td>Ultra-microtome</td><td>Leica</td><td>UCT</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400</td><td>Hazardous chemical</td></tr><tr><td>Lead citrate</td><td>EMS</td><td>17900</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>Hitachi</td><td>H7600</td><td></td></tr><tr><td>Nickel grid</td><td>EMS</td><td>G200-Ni</td><td></td></tr><tr><td>Copper grid</td><td>EMS</td><td>G200-Cu</td><td></td></tr><tr><td>Transmission Electron Microscopy</td><td>JEOL</td><td>JEM2200FS</td><td></td></tr></tbody></table>

sectioning, staining and tem imaging of embedded exosomes: a protocol to visualize the structural features of exosomes using tem

Source: Jung, M. K. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/56482" target="_blank">Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.</a> J. Vis. Exp. (2018)
This video describes the technique for preparing a thin section of embedded exosomes. These sections can further be stained and imaged using transmission electron microscopy, or TEM, to study the structural features of exosomes.

Sectioning, Staining and TEM Imaging of Embedded Exosomes: A Protocol To Visualize the Structural Features of Exosomes Using TEM

Source: Jung, M. K. et al. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)
This video describes the ...

Exosomes are nanosized, spherical vesicles enclosed by a lipid bilayer containing DNA, RNA, and protein molecules present inside the lumen.
To visualize the exosome structure using transmission electron microscopy, or TEM, begin by taking an exosome block - a solid matrix block containing the embedded exosomes. Using an ultra-microtome, obtain ultrathin sections of the exosome block.
Collect the exosome section on a suitable grid and allow it to adhere to the surface. The grid provides stable support to the exosome specimen for subsequent staining steps.
Next, position the grid over a drop of uranyl acetate - a heavy metal stain - and incubate for the desired duration. Uranyl ions interact with lipids, proteins, DNAs, and RNAs present in the exosomes.
Subsequently, stain the exosome section with lead citrate, which also binds to the exosomal biomolecules. Lead citrate weakly binds to the pre-bound uranyl ions and enhances the contrast against background during imaging.
Observe the doubly stained exosomes using TEM. TEM focuses the electron beam on the exosome sample. Stained regions in the exosome scatter electrons more strongly in comparison to the unstained regions.
The objective aperture filters these highly scattered electrons. Thus, exosomes containing stained biomolecules appear darker than the background.

sectioning-staining-tem-imaging-embedded-exosomes-protocol-to

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

1.4K 次观看. 来源:Jung， M. K. et al. 通过透射电子显微镜对外泌体进行样品制备和成像。J. Vis. Exp.（2018 年） 该视频描述了制备包埋外泌体薄片的技术。这些切片可以使用透射电子显微镜 （TEM） 进一步染色和成像，以研究外泌体的结构特征。 

视频: 嵌入外泌体的切片、染色和 TEM 成像:使用 TEM 可视化外泌体结构特征的方案 - 概念

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

一种在透射电镜中获取微生物连续超薄切片的方法

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

科研

JoVE Journal

工程学

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

微生物菌落生物膜的石蜡嵌入和薄切片显微分析

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

免疫与感染

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

切片中石蜡嵌入组织的切割和漂浮方法

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

Sectioning, Staining and TEM Imaging of Embedded Exosomes: A Protocol To Visualize the Structural Features of Exosomes Using TEM

成績單

Sectioning, Staining and TEM Imaging of Embedded Exosomes: A Protocol To Visualize the Structural Features of Exosomes Using TEM