Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF5 Cites 8 Cited by
  • 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, and it is necessary to establish a Fluorescence double-labeled microscopic observation method of tubulin and DNA during the early embryonic development of fertilized eggs of marine flounder and flounder

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N33/68G01N33/577G01N33/531G01N33/533
Inventor 朱香萍林正美尤锋吴志昊
Owner QINGDAO AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com