A Method for Extracting Nuclei from Wheat Roots Suitable for Immunofluorescence Analysis

A technology of immunofluorescence and extraction method, which is applied in the field of cell biology, can solve the problems of not being suitable for wheat crops, etc., and achieve the effect of good staining effect, maintaining activity and increasing efficiency

Active Publication Date: 2021-03-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the different structures and tissue sources of different plants, the preparation method suitable for other plant nuclei is not suitable for wheat crops

Method used

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  • A Method for Extracting Nuclei from Wheat Roots Suitable for Immunofluorescence Analysis
  • A Method for Extracting Nuclei from Wheat Roots Suitable for Immunofluorescence Analysis
  • A Method for Extracting Nuclei from Wheat Roots Suitable for Immunofluorescence Analysis

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Experimental program
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Effect test

Embodiment 1

[0038] A method for extracting cell nuclei from wheat roots suitable for immunofluorescence analysis, the steps of which are:

[0039] (1) Obtaining of young wheat roots:

[0040] The experimental material is the wheat variety (Triticum aestivum L.) Huamai No. 8. After the seeds are disinfected and washed, they are placed in a petri dish with moist filter paper to germinate in the dark (20-25°C), and the seedlings are moved to the culture room for growth after 1 day of germination. During the growth period, the nutrient solution was replenished twice. After the seedlings grew to the fifth day, the wheat radicles were taken for the cell nucleus extraction experiment. The cultivation conditions of the seedlings in the cultivation room: 18-23°C, 65-75% relative humidity, 12 hours of light -14h / d.

[0041] Described nutrient solution is 1 / 2 Hoagland's nutrient solution, and its formula is: 2mM calcium nitrate (Ca(NO 3 ) 2 ), 2.5mM potassium nitrate (KNO 3 ), 0.5mM ammonium nit...

Embodiment 2

[0061] Optimization and Improvement of Nuclei Filter Method

[0062] The method of cell nucleus filtration is a key experiment in the preparation process of cell nucleus suspension. The improvement and optimization of the extraction method of wheat root nucleus in this experiment is mainly aimed at the improvement of the complicated and inconvenient cell nucleus filtration method in the past. First of all, the filter equipment consists of the cell mesh screen ( figure 1 A) Replaced by a simple homemade filter ( figure 1 C), the required material is a 450 mesh nylon membrane and a sterilized 1.5ml centrifuge tube ( figure 1 B) After cutting the nylon membrane into a disc with a diameter of 2cm, fold it into a hole shape and put it into the centrifuge tube. The three-layer filter membrane is close to the tube wall ( figure 1 C).

[0063] Compared with the extraction of plant tissue nuclei, the current existing cell filter screens are relatively large in size and cannot be...

Embodiment 3

[0066] Comparison of Three Different Extraction Methods for Extracting Nuclei from Wheat Young Roots

[0067] The results of extracting wheat radicle nuclei by different methods are as follows: figure 2 shown. It can be seen from the figure that there are many impurities on the cell nucleus glass slide prepared by the shearing method, and it is difficult to distinguish cell fragments from the nucleus ( figure 2 A). In addition, some cell nuclei are incomplete in shape, and the nuclei are mostly in clusters, which are difficult to separate when they stick together ( figure 2 A1). However, the number of nuclei in the nucleus suspension obtained by the shearing method was the largest ( figure 2 D).

[0068] The preparation of cell nucleus suspension by enzymatic hydrolysis takes a long period of time, and it is not suitable to ensure the activity of cell nucleus. Moreover, the method extracts the least number of nuclei ( figure 2 D). However, the nuclei prepared by this m...

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Abstract

The invention discloses a wheat root cell nucleus extraction method applicable to immunofluorescence analysis. The wheat root cell nucleus extraction method comprises the following steps: (1) obtaining wheat young roots; (2) extracting cell nucleuses; (3) detecting the cell nucleuses through a DAPI (Diamidino-phenyl-indole) fluorescent staining method; (4) carrying out immunofluorescence histochemical analysis; (5) carrying out statistic analysis. According to the cell nucleus extraction method provided by the invention, the time for extracting the cell nucleuses can be greatly reduced and theactivity of protein in the cell nucleuses is kept for a longer time; the accuracy and success rate of subsequent target protein detection are improved, the concentration of impurities in a cell nucleus suspension solution is reduced, influences, caused by the impurities, in an immunofluorescence assay are reduced, the quantity of the cell nucleuses in the cell nucleus suspension solution is increased and related statistical experiment and analysis are convenient to carry out subsequently. The extraction method provided by the invention is applicable to production of the cell nucleus suspension solution so that subsequent submicroscopic-level cytological related experiments are convenient to carry out.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for extracting cell nuclei from wheat roots suitable for immunofluorescence analysis, and the method is suitable for preparing cell nucleus suspensions for subsequent submicroscopic cytology-related experiments. Background technique [0002] Immunofluorescence technology (immunofluorescence) is based on the principle of antigen-antibody recognition reaction. First, known antigens or antibodies are labeled with fluorescein to make fluorescent markers, and then this fluorescent antibody (or antigen) is used as a molecular probe to examine cells or The corresponding antigen (or antibody) in the tissue. The antigen-antibody complexes formed in cells or tissues contain fluorescein, and fluorescence-labeled cells or tissues can be observed with a fluorescence microscope to determine the nature and location of antigens or antibodies, or to conduct quantitative ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/30G01N33/533
CPCG01N1/30G01N33/533
Inventor 李柽钖周竹青
Owner HUAZHONG AGRI UNIV
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