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A method for eliminating autofluorescence in plant tissue

A plant tissue and autofluorescence technology, applied in the field of fluorescence detection, can solve the problems of difficulty in purchasing, lack of relevant instruments, and high hardware requirements

Active Publication Date: 2022-07-08
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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  • A method for eliminating autofluorescence in plant tissue
  • A method for eliminating autofluorescence in plant tissue
  • A method for eliminating autofluorescence in plant tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Include the following steps:

[0032] 1. Astragalus seed hypocotyl is the test material, sliced ​​by hand.

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

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

[0035] 4. Observe the degree of fluorescence quenching of slices with different soaking time 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 30mg / ml ninhydrin solution (solvent is distilled water) for 15min.

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

[0040] The result is as figure 2 shown.

Embodiment 3

[0042] Include the following steps:

[0043] 1. Take the embryonic axis of Astragalus seeds as the test material, and slice them by hand.

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

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

[0046] 4. Label the nuclei of the tissue sections with two 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 for five minutes each time.

[0047] 5. Observe and photograph the sections under a biofluorescence microscope. The 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 tissue. In the method, the slice of plant tissue is incubated in a ninhydrin solution, which can effectively remove the interference of autofluorescence, improve the signal-to-noise ratio of the image, and make the specific fluorescence clearly visible, which is simple, fast, 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 tissue. Background technique [0002] With the rapid development of life science research, bioluminescence technology has become more and more widely used in cellular immunology, microbiology, molecular biology, molecular genetics, medicine, and botany. Fluorescence technology has become one of the important means of life science research. [0003] However, some molecules in cells can fluoresce when exposed to ultraviolet light or a certain wavelength of visible light. This fluorescence is caused by molecules endogenous to the cell, so it is called auto-fluorescence. For example, there are tryptophans, indoleamines, fibrinogen, dimer, collagen, etc. excited in the ultraviolet and violet regions, and NADH, lipofuscin, indoleamines, dimers, and riboflavin are excited in the blue light region. white. [0004] Auto...

Claims

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Application Information

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Patent Type & Authority Patents(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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