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Method for preparing scanning electron microscope samples from biological samples

A biological sample, scanning electron microscope technology, applied in the preparation of test samples, etc., can solve the problems of low accuracy, different first protein and second protein, etc.

Inactive Publication Date: 2014-09-24
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the shortcomings of the low accuracy of observation in low vacuum mode in the existing methods for preparing scanning electron microscope samples, the present invention provides a method for preparing biological samples into scanning electron microscope samples, the biological samples contain the first protein and a second protein, the first protein and the second protein being different; the method comprising the steps of:

Method used

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  • Method for preparing scanning electron microscope samples from biological samples
  • Method for preparing scanning electron microscope samples from biological samples
  • Method for preparing scanning electron microscope samples from biological samples

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Experimental program
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Embodiment approach

[0026] According to a preferred embodiment of the method according to the invention, said first contacting is carried out in the presence of bovine serum albumin at a concentration of 0.5-2% by weight.

[0027] According to the method of the present invention, wherein, the conditions of the second contact are not particularly required, and can be conventional conditions of immunoelectron microscopy, for example (The Journal of Cell Biology (The Journal of Cell Biology), 1984, volume 99, page 53 ) conditions described in the literature, in order to make the prepared scanning electron microscope sample can accurately distinguish two different protein molecules, preferably, the conditions of the second contact include: relative to each mg of biological sample, labeled with The amount of the first antibody of the first gold nanoparticle or the second antibody labeled with the second gold nanoparticle is 100-800 micrograms, more preferably 200-600 micrograms; the contact temperature...

preparation example 1

[0063] This preparation example is used to illustrate the method for preparing the first antibody labeled with the first gold nanoparticles and the second antibody labeled with the second gold nanoparticles.

[0064] 1. Prepare the following solution:

[0065] (1) 0.1M cetyltrimethylammonium bromide (C 19 h 42 N + Br - , hereinafter referred to as CTAB) aqueous solution;

[0066] Weigh 3.64g of cetyltrimethylammonium bromide (analytical grade) and dissolve in 100mL of deionized water; before use, it should be placed in a constant temperature water bath at 30°C to dissolve completely to obtain 0.1M cetyltrimethylammonium bromide ammonium bromide (C 19 h 42 N + Br - , hereinafter referred to as CTAB) aqueous solution;

[0067] (2) 0.1M aqueous ascorbic acid (AA) aqueous solution:

[0068]Weigh 0.176g of ascorbic acid (analytical pure) and dissolve it in 10mL of deionized water to obtain a 0.1M ascorbic acid (AA) aqueous solution (the solution can be temporarily prepare...

preparation example 2

[0099] This preparation example is used to illustrate the preparation method of the biological material mentioned in the present invention.

[0100] The preparation purpose of this preparation example is to prepare rat brain microvascular endothelial cells stimulated by burn serum, and the steps are as follows:

[0101] (1) Primary culture of rat brain microvascular endothelial cells:

[0102] Adult Wistar male rats were anesthetized with 3.5% by weight of chloral hydrate and then decapitated to take out the brain tissue, which was placed in a petri dish; the pia mater, large blood vessels and white matter were removed to obtain the cerebral cortex, and the obtained cerebral cortex was cut After crushing, use Dounce (Dounce) homogenizer to homogenate to obtain a homogenate, and filter the homogenate with a 180-mesh filter to obtain a filtrate; Digest at 37°C for 15 minutes to obtain a digestive solution; centrifuge the digestive solution at a speed of 800g for 5 minutes and d...

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Abstract

The invention provides a method for preparing scanning electron microscope samples from biological samples. The method comprises enabling a first antibody marked with first gold nanoparticles and a second antibody marked with second gold nanoparticles to contact with a biological sample after the biological sample is in resistance treatment; and then successively performing fixation, gradient dehydration and critical point drying. The shapes of the first gold nanoparticles are different from those of the second gold nanoparticles, and the first gold nanoparticles and the second gold nanoparticles are one or more from cylindrical gold nano bars, octahedral gold nanoparticles, spherical gold nanoparticles and arrow-shaped gold nanoparticles. The scanning electron microscope samples prepared by the method can accurately distinguish two types of different protein molecules in a low-vacuum mode.

Description

technical field [0001] The invention relates to a method for preparing electron microscope samples, in particular to a method for preparing biological samples into scanning electron microscope samples. Background technique [0002] Scanning electron microscopy imaging of protein molecules in biological samples can be achieved by using appropriate markers. This nanoscale high-resolution imaging can provide a powerful tool for better analysis of the distribution and interaction of protein molecules in biological samples. The preparation of SEM samples with selected markers is critical for the effect of SEM imaging based on immune responses. [0003] As a marker for scanning electron microscopy, it needs to have the following characteristics: (1) has good secondary electron imaging (2) has less non-specific adsorption with samples (3) is easy to bind to antibodies through specific modification (4) in Stable in physiological environment. Due to its controllable morphology, goo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28
Inventor 张珂吴晓春韩东
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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