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Method for producing biolgoical sample embedded block based on atomic force microscope observation

An atomic force microscope and biological sample technology, applied in the field of bioengineering, can solve the problems of expensive, cumbersome steps, long time-consuming, etc., and achieve the effects of shortening sample preparation time, simple method steps, and improving resolution

Inactive Publication Date: 2006-05-17
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, this method developed for electron microscope research may not be the most suitable for AFM to study the fine structure of biological samples, and there are many problems in it, for example: (1) Osmium tetroxide cannot improve the contrast of AFM images, And it is expensive and has certain toxicity; (2) Osmium tetroxide can undergo oxidation-reduction reaction with ethanol and glutaraldehyde to produce precipitation, which has an adverse effect on the ultrastructure of cells observed by AFM; (3) The steps of this method are relatively cumbersome, It takes a long time, and ordinary technicians need professional training to master it, so it is not suitable for promotion and use

Method used

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  • Method for producing biolgoical sample embedded block based on atomic force microscope observation
  • Method for producing biolgoical sample embedded block based on atomic force microscope observation
  • Method for producing biolgoical sample embedded block based on atomic force microscope observation

Examples

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Effect test

Embodiment 1

[0018] Human tongue squamous cell carcinoma tissue samples were taken from the clinical operation of oral and maxillofacial surgery in a hospital, cut into small pieces less than 2 mm square, and fixed with 2% glutaraldehyde fixative solution pre-cooled in ice water for 2 hours. The fixative formula is 50ml of 0.2mol / L phosphate buffer solution, 8ml of 25% glutaraldehyde aqueous solution, add ultrapure water to 100ml, and put it in an ice-water bath to cool it for later use. Then wash twice with 0.2mol / L phosphate buffer, and then carry out ethanol gradient dehydration. The dehydration process is as follows:

[0019] Under freezing conditions (0°C-4°C), 50% ethanol water solution for 15 minutes, 70% ethanol water solution for 15 minutes, and 95% ethanol water solution for 15 minutes at room temperature, anhydrous ethanol was replaced once, each time 10 minutes Minutes, absolute ethanol was replaced once, every 10 minutes.

[0020] After the sample has been dehydrated, it is d...

Embodiment 2

[0023]After culturing C6 cells, pour out the culture medium, first wash twice with 0.2mol / L phosphate buffer solution, then add ice water pre-cooled 2% glutaraldehyde fixative solution to fix at low temperature for 1 hour, and centrifuge the obtained cell mass with ethanol The solution is subjected to gradient dehydration, followed by 50%→70%→95%→absolute ethanol→absolute ethanol, 10 minutes each time, in which absolute ethanol and absolute ethanol should be replaced once respectively, and ice water is required for 50% and 70% ethanol Pre-chill and dehydrate at low temperature. After the sample was dehydrated, it was directly transferred to the liquid with a volume ratio of epoxy resin 618: absolute ethanol = 1:1 and placed on a shaker to infiltrate for 1 hour, then poured out the liquid, blotted the residue with filter paper, and then added ring Oxygen resin 618 mixed solution, infiltrated in a 35°C incubator for 3 hours. Shake a few times in the middle to facilitate penetra...

Embodiment 3

[0026] After Tca8113 cells are cultured, discard the culture medium, rinse with 0.2mol / L phosphate buffer, add ice water pre-cooled 2% glutaraldehyde fixative solution to fix at low temperature for 1.5 hours, and dehydrate the cell mass obtained after centrifugation with gradient ethanol solution , followed by 50%→70%→95%→absolute ethanol→absolute ethanol, every 12 minutes, anhydrous and absolute ethanol are replaced once, 50% and 70% ethanol need to be pre-cooled with ice water and carried out at low temperature dehydration. After the sample is dehydrated, directly use the epoxy resin 618 mixed solution: absolute ethanol = 1:1 volume ratio liquid to infiltrate on the shaker for 1.5 hours, then pour the solution, absorb the residue with filter paper, and then add epoxy Resin 618 mixed solution, infiltrated in an incubator at 35°C for 3.5 hours. Pick out the sample, carefully absorb the residue with filter paper, and embed it with epoxy resin 618 mixture. Then heat polymeriza...

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Abstract

The invention discloses a preparation method of a biological sample embedding block based on atomic force microscope observation, which belongs to the technical field of bioengineering. Use 2% glutaraldehyde fixative to fix biological tissue samples or cell samples at low temperature, wash with 0.2mol / L phosphate buffer, follow the ethanol gradient of 50% → 70% → 95% → absolute ethanol → absolute ethanol Carry out dehydration, embedding after infiltration by epoxy resin 618, and then heating and polymerizing. The heating temperature and time are: 35°C for 12 hours → 45°C for 12 hours → 60°C for 24 hours. Thus, a biological sample embedding block suitable for AFM observation is obtained. The steps of the method of the invention are relatively simple, most of the reagents used are domestic, easy to obtain and low in price, and are suitable for making embedding blocks of various animal tissues and cell samples.

Description

technical field [0001] The invention relates to a method for preparing a biological sample, in particular to a method for preparing an embedding block of a biological sample based on atomic force microscope observation, and belongs to the technical field of bioengineering. Background technique [0002] After the invention of the atomic force microscope (AFM) in the 1980s, it has been used more and more in the biological field, many of which use AFM to observe the fine structure of ultra-thin sections of biological samples. Earlier literature can be found in "Imaging of the Surface Structures of EponThin Sections Created with a Glass Knife and a Diamond Knife by the Atomic Force Microscope" (Amako K, Takade A, Umeda A et al. J Electron Microsc. 1993, 42( 1993) 121-123), "Atomic force microscopy of embedding-free sections of cells and tissues" (Ushiki T, Shigeno M & Abe K. Arch Histol Cytol.1994, 57(1994): 427-432), they tried to use AFM To observe the ultra-thin sections for...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N1/42G01N1/44
Inventor 李鑫辉季彤胡晓芳刘苹张晓东胡钧
Owner SHANGHAI JIAO TONG UNIV
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