Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses

A technique for microscopic observation of fertilized eggs, applied in the fields of immunocytochemistry and fluorescence microscopy, which can solve problems such as application limitations and achieve a wide range of applications

Inactive Publication Date: 2013-10-23
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the application of the conventional fluorescent double-labeled microscopic observation technique of tubulin and DNA in the early embryonic development of Homoyellow zygote (mammalian) is limited

Method used

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  • Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses
  • Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses
  • Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Fluorescence double-labeled microscopic observation method of turbot 1-cell interphase zygote tubulin and nuclear DNA

[0047] 1. The sample was fixed with formaldehyde-glutaraldehyde fixative solution prepared by Pipes. After the unmembraned turbot fertilized eggs were incubated in the fixative solution for 3 hours at room temperature, they were directly treated with phosphate buffered Tween solution (PBST) (PBST: 128 mM NaCl, 2 mM KCl, 8 mM NaH 2 PO 4 , 2 mM KH 2 PO 4 , 0.1% Tween-20, pH 7.2) to replace the corresponding volume of fixative and store at 4°C until use.

[0048] 2. Under the dissecting microscope, the fertilized eggs with normal development status are selected, and the blastodisc is separated from the whole fertilized eggs with a dissecting needle.

[0049] 3. Punch the sample with 4% Triton-100 at 25°C for 12 hours. Then use 2.5mg / L sodium borohydride (NaBH 4 ) solution to nurture the sample three times, each time no more than half an ...

Embodiment 2

[0057] Example 2, Fluorescent double-labeled microscopic observation process of turbot 2-cell metaphase zygote tubulin and nuclear DNA

[0058] The operating steps are basically the same as in Example 1, and the differences from Example 1 are:

[0059] The fertilized eggs of turbot were reared in formaldehyde-glutaraldehyde fixation solution for 6 hours;

[0060] The development stage is 2-cell metaphase, the temperature of Triton-100 punching treatment is 30°C, the concentration is 5%, and the treatment time is 12 hours;

[0061] The dilution ratio of monoclonal mouse anti-α-tubulin (α-Tubulin) is 1:750;

[0062] The dilution ratio of goat anti-mouse IgG (H+L) labeled with Alexa Fluor 555 is 1:500;

[0063] DAPI dilution ratio is 1:10000.

Embodiment 3

[0064] Example 3, Fluorescent double-labeled microscopic observation process of turbot 4-cell metaphase zygote tubulin and nuclear DNA

[0065] The operating steps are basically the same as in Example 1, and the differences from Example 1 are:

[0066] Fertilized turbot eggs were reared in formaldehyde-glutaraldehyde fixative for 5 hours;

[0067] The development stage is 4-cell metaphase, the temperature of Triton-100 punching treatment is 28°C, the concentration is 4%, and the treatment time is 10 hours;

[0068] The dilution ratio of monoclonal mouse anti-α-tubulin (α-Tubulin) is 1:500;

[0069] The dilution ratio of goat anti-mouse IgG (H+L) labeled with Alexa Fluor 555 is 1:250;

[0070] DAPI dilution ratio is 1:10000.

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Abstract

The invention discloses a method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses. The method comprises the following steps of carrying out fixing of an oosperm with a membrane, stripping a blastoderm of the fixed oosperm from a whole embryo by a dissecting needle, punching the stripped blastoderm, reducing or eliminating the influence produced by uncombined aldehyde groups on an experimental result by sodium borohydride, carrying out sample sealing and fluorescent double-labeling dyeing, and carrying out sample observation by a fluorescence microscope or a laser scanning confocal microscope. The method has low sample requirements, has simple processes, is convenient for observation, produces a clear result, has a high three-dimensional feel, can be used for dynamic change research on flounder oosperm microtubules and cell nucleuses from blastoderm formation respectively to 2-cell, 4-cell and 8-cell development stages, is suitable for other larger telolecithal eggs of marine flounder oosperms, and has a wide application range.

Description

technical field [0001] The invention relates to immunocytochemical technology and fluorescent microscopic technology, in particular to a microscopic observation method for fluorescent double labeling of the microtubule skeleton and nucleus of fertilized eggs of marine flounder and flounder. Background technique [0002] Immunocytochemistry is a technique based on the principle of immunology, which uses the combination of antibodies and specific antigens to determine the specific location of antigens. If the antibody is bound to a marker and then reacts with the antigen in the tissue, the site where the antigen exists in the tissue can be displayed under a light microscope or an electron microscope. Therefore, this technique is a general term for some research methods that combine specific reactions between antigens, antibodies, and complements in immunology with microscopic or submicroscopic histology. It is a combination of immunological principles and light microscopy or e...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/531G01N33/533
Inventor 朱香萍林正美尤锋吴志昊
Owner QINGDAO AGRI UNIV
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