Immune fluorescent marking method for naked plant pollen tube microtubule skeleton and its use

A technique of gymnosperm and immunofluorescence, which is applied in the field of immunofluorescence labeling of the microtubule skeleton of gymnosperm pollen tubes, can solve the problems of lagging biological research, achieve good labeling effect, omit the sealing step, and have high application value

Inactive Publication Date: 2007-11-21
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no effective methods and techniques for labeling microtubule skeletons in gymnosperm pollen grains and pollen tubes have been reported, resulting in a lag in biological research related to gymnosperms compared to angiosperms

Method used

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  • Immune fluorescent marking method for naked plant pollen tube microtubule skeleton and its use
  • Immune fluorescent marking method for naked plant pollen tube microtubule skeleton and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Immunofluorescent labeling of the microtubule skeleton of white rod pollen tubes and its microscopic observation

[0033] Using the method of the present invention to carry out immunofluorescent labeling on the microtubule skeleton of the white rod pollen tube, comprising the following steps:

[0034] 1) collect the white rod pollen tube sample to be marked, in freshly prepared 50mMPipes damping solution containing 4% (g / ml) paraformaldehyde (every liter of water contains 50mM PIPES, 2mM MgCl 2 , 5mM EGTA, pH 6.9) for 60min by fast pumping, the amount of buffer is based on the soaked pollen tube sample;

[0035] 2) wash with 50mM Pipes buffer 3 times, each time for 10min, to remove residual paraformaldehyde;

[0036] 3) Put the pollen tube in 1g / 100ml of pectinase (purchased from Yakult Honsha Co.Ltd, 3000U / g) and cellulase (purchased from Yakult Honsha Co.Ltd, 3000U / g) solution at 25°C Enzymolysis for 35 minutes, wherein the weight-number ratio of pectinas...

Embodiment 2

[0044] Example 2. Fluorescence labeling of the microtubule skeleton of the pollen tubes of Pythias lanceolata and its microscopic observation

[0045]Using the method of the present invention to carry out immunofluorescent labeling on the microtubule skeleton of the blue pole pollen tube, comprising the following steps:

[0046] 1) collect the blue stem pollen tube sample to be marked, in freshly prepared 60mMPipes buffer containing 3% (g / ml) paraformaldehyde (every liter of water contains 60mM PIPES, 2mM MgCl 2 , 5mM EGTA, pH 6.9) for 60min by fast pumping, the amount of buffer is based on the soaked pollen tube sample;

[0047] 2) wash with 40mM Pipes buffer 4 times, each time for 5min, to remove residual paraformaldehyde;

[0048] 3) Place the pollen tube in 1g / 100ml of pectinase (purchased from Yakult Honsha Co.Ltd, 3000U / g) and cellulase (purchased from Yakult Honsha Co.Ltd, 3000U / g) solution at 30°C Enzymolysis for 30 minutes, wherein the weight-number ratio of pectina...

Embodiment 3

[0056] Example 3. Fluorescent labeling of the microtubule skeleton of Pinus tabulaeformis and its microscopic observation

[0057] Using the method of the present invention to carry out immunofluorescent labeling on the microtubule skeleton of Pinus tabulaeformis tubes, comprising the following steps:

[0058] 1) collect the pine pollen tube sample to be marked, in freshly prepared 40mMPipes damping solution containing 5% (g / ml) paraformaldehyde (every liter of water contains 40mM PIPES, 2mM MgCl 2 , 5mM EGTA, pH 6.9) and quickly pumped and fixed for 50min, the amount of buffer was based on the soaked pollen tube sample;

[0059] 2) wash with 60mM Pipes buffer twice, each time for 15min, to remove residual paraformaldehyde;

[0060] 3) Put the pollen tube in 0.5% (W / W) pectinase (purchased from Yakult Honsha Co.Ltd, 3000U / g) and cellulase (purchased from Yakult Honsha Co.Ltd, 3000U / g) solution enzymatic hydrolysis at 25°C for 40 minutes, wherein the weight-number ratio of pe...

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Abstract

An immune-fluorescence labeling method of micro-pipe frame at pollen tube on gymnosperm includes fixing pollen tube of gymnosperm on fixing liquid for 40-60min, washing pollen tube, placing pollen tube in enzyme solution for carrying out enzymolysis of 30-40 min under temperature of 20-30deg.c, washing pollen tube, setting pollen tube in Triton X-100 to penetrate there for 1-3h, washing pollen tube, adding anti-beta-tubulin single clone antibody to carry out hatching for 1-3h, washing pollen tube, adding fluorescence label to carry out shielded hatching for 1-3h for making micro-pipe frame be immune-fluorescence labeled.

Description

technical field [0001] The present invention relates to the immunofluorescent labeling method of pollen tube microtubule skeleton and its application in the field of plant biotechnology, in particular to a method for immunofluorescent labeling of gymnosperm pollen tube microtubule skeleton and the use of the method for gymnosperm pollen pollen Applications of Microtubule Skeleton for Microscopy. Background technique [0002] As the carrier of the male reproductive unit in the fertilization process of flowering plants, pollen tube has typical apical growth characteristics, so it has become an ideal model system for studying the growth of cell polarity in the field of biotechnology. In addition, the pollen tube system is also of great significance to the study of the interaction between cells and the study of signal transduction. The cytoskeleton is the basic organizational structure of the pollen tube, and it participates in important physiological processes such as cell sha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N21/64G01N21/84
Inventor 林金星王晓华盛仙永
Owner INST OF BOTANY CHINESE ACAD OF SCI
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