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Method for eliminating autofluorescence of plant tissue

A plant tissue and autofluorescence technology, applied in the field of fluorescence detection, can solve the problems of background fluorescence enhancement, interference with specific fluorescent labels, and difficulty in purchasing

Active Publication Date: 2021-06-08
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the photobleaching technology requires high hardware conditions, the general laboratory lacks relevant instruments, and the tissue damage caused by photobleaching is irreversible
The chemical reagents mainly used in the chemical treatment method are dyes such as sodium borohydride, trypan blue and Sudan black. Sodium borohydride is a flammable and explosive substance, which is not easy to purchase and store, and the reproducibility of the use effect is low.
Dyes such as trypan blue and Sudan black may cause uneven staining and enhanced background fluorescence, and the overall reduction in autofluorescence is not large, and still interfere with specific fluorescent labeling

Method used

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  • Method for eliminating autofluorescence of plant tissue
  • Method for eliminating autofluorescence of plant tissue
  • Method for eliminating autofluorescence of plant tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Include the following steps:

[0032] 1. The hypocotyl of Astragalus membranaceus seeds is used as the test material, and sliced ​​by hand.

[0033] 2. The slices were soaked in 20 mg / ml ninhydrin solution (1×PBS buffer solution with pH 7.4) for different time (0 min, 2 min, 10 min, 20 min).

[0034] 3. Rinse twice with PBS after soaking.

[0035] 4. Observe the degree of fluorescence quenching of slices with different soaking times under a fluorescence microscope.

[0036] The result is as figure 1 shown.

Embodiment 2

[0038] 1. Take the roots of Arabidopsis thaliana and soak them in 30 mg / ml ninhydrin solution (distilled water as the solvent) for 15 minutes.

[0039] 2. Observe the quenching of root autofluorescence under a fluorescence microscope

[0040] The result is as figure 2 shown.

Embodiment 3

[0042] Include the following steps:

[0043] 1. Take the hypocotyl of the Astragalus membranaceus seed as the test material, and slice it by hand.

[0044] 2. The slices were permeabilized with 0.3% Triton X-100 solution for 20 minutes, washed twice with PBS, five minutes each time.

[0045] 3. Then incubate with ninhydrin solution (1×PBS with pH 7.4 as solvent, the concentration is 20mg / ml), add the solution to the EP tube with slices, cover the slices, about 10-20 minutes, Carry out at room temperature, and observe that the slices are stained blue-purple. Rinse twice with PBS, five minutes each time.

[0046] 4. Tissue slices were labeled with two kinds of fluorescent probes at the same time, namely EasyProbes TM Green 488Live Cell Stain (60μl / 100ml), PI (6μg / ml), reaction in the dark for 15min, washed twice with PBS, five minutes each time.

[0047] 5. The slices were observed and photographed under a biofluorescence microscope. Excitation and emission wavelengths are:;...

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Abstract

The invention relates to the technical field of fluorescence detection, in particular to a method for eliminating autofluorescence of plant tissues. According to the method, a plant tissue slice is placed in the ninhydrin solution for incubation treatment, so that the interference of autofluorescence can be effectively removed, the signal-to-noise ratio of an image is improved, and the specific fluorescence is clear, visible, simple, rapid, safe and efficient.

Description

technical field [0001] The invention relates to the technical field of fluorescence detection, in particular to a method for eliminating autofluorescence of plant tissues. Background technique [0002] With the rapid development of life science research, bioluminescence technology is more and more widely used in cellular immunology, microbiology, molecular biology, molecular genetics, medicine, botany, etc. Fluorescence technology has become one of the important means of life science research. [0003] However, certain molecules in cells can fluoresce when exposed to ultraviolet light or certain visible light bands. This fluorescence is caused by endogenous molecules in the cell, so it is called auto-fluorescence. For example, tryptophans, indoleamines, fibrinogen, dimers, collagen, etc. are excited by ultraviolet and purple light regions, and NADH, lipofuscin, indoleamines, dimers, and riboflavin are mainly excited by blue light regions. white. [0004] Autofluorescence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 郭靖鲁海坤于营张浩闫梅霞隋昕刘亚苓
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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