Immunofluorescence microscopy observation method for marine bivalve meiosis device

A technique of meiosis and immunofluorescence, applied in the field of cytochemistry and cellular immunology, can solve the problems such as hindering further research on fertilization mechanism, unable to reveal the interaction between spindle and chromosome, etc. Simple operation effect

Inactive Publication Date: 2010-02-03
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] To sum up, most of the fluorescent staining methods used in the reported seawater bivalves fertilization biology research are to observe a part of the meiotic apparatus (spindle or chromosome), which splits the biological process of meiosis. The inability to reveal the interaction between the spindle and chromosomes hinders further research on the mechanism of fertilization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Utilize the method of the present invention, carry out long oyster zygote meiotic organ immunofluorescence microscopic observation,

[0021] The specific operation is:

[0022] 1) Filter through a sieve with a pore size of 25 μm to remove most of the seawater, obtain about 10,000 fertilized oyster eggs at a specific developmental stage, put them in a 1.5mL centrifuge tube, and centrifuge at 2000rpm for 4min to remove the supernatant seawater; add about 1mL After 10 min of fixative, centrifuge at 2000 rpm for 4 min, remove the supernatant, collect fertilized eggs, add 1 mL of fixative to fix for 30 min, and set aside. The fixative is 2% paraformaldehyde in phosphate buffer.

[0023] 2) Take about 60 fertilized eggs and transfer them into an incubation solution for permeabilization for 30 minutes; the incubation solution is a PBS solution of polyethylene glycol octylphenyl ether (Triton X-100) with a concentration of 0.5% by volume.

[0024] 3) The incubation solution w...

Embodiment 2

[0031] The difference from Example 1 is that the immunofluorescent microscopic observation of the fertilized egg mitotic organ of Chlamys farreri is carried out, and the specific operations are as follows:

[0032] 1) Filter through a sieve with a pore size of 30 μm to remove most of the seawater, and obtain about 8,000 fertilized eggs of Chlamys farreri at a specific developmental stage, put them in a 1.5mL centrifuge tube, and centrifuge at 1,000rpm for 5 minutes to remove the supernatant seawater; add After about 1 mL of fixative for 10 min, centrifuge at 1000 rpm for 5 min, remove the supernatant to collect fertilized eggs, then add 1 mL of fixative to fix for 60 min, and set aside. The fixative is 4% paraformaldehyde in phosphate buffer.

[0033] 2) Take about 30 fertilized eggs and transfer them to an incubation solution for permeabilization for 40 minutes; the incubation solution is a PBS solution of polyethylene glycol octylphenyl ether (Triton X-100) with a concentrat...

Embodiment 3

[0040] The difference from Example 1 is that the immunofluorescent microscopic observation of the fertilized egg mitotic organ of the Philippine clam is carried out, and the specific operation is as follows:

[0041] 1) Filter through a sieve with a pore size of 30 μm to remove most of the seawater, obtain about 5,000 fertilized eggs of Philippine clams at a specific development stage, put them in a 1.5mL centrifuge tube, and centrifuge at 1,000rpm for 5 minutes to remove the supernatant seawater; add After about 1mL of fixative for 10min, centrifuge at 1000rpm for 5min, remove the supernatant to collect fertilized eggs, then add 1mL of fixative to fix for 50min, and set aside. The fixative is 6% paraformaldehyde in phosphate buffer.

[0042] 2) Take about 40 fertilized eggs, and permeabilize the incubation solution for 60 minutes. The incubation solution is a PBS solution of polyethylene glycol octylphenyl ether (Triton X-100) with a concentration of 0.8% by volume.

[0043]...

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Abstract

The invention relates to cytochemistry and cellular immunology, which are particularly used for observing a marine bivalve meiosis device through immunofluorescence microscopy. A sample can be observed after pre-treatment of fixing and the like, dyeing and flaking. The sample is fixed by phosphate buffered saline (PBS) of paraformaldehyde at a mass percentage concentration of 2 to 6 percent; then,permeabilization treatment and sealing treatment are performed sequentially; immunofluorescence dyeing is performed on sample spindle bodies, and fluorescence dyeing is performed on chromosomes withthe phosphate buffered saline containing propidium iodide of which the concentration is 5 to 20 mg of per liter; and the sample is flaked after dyeing for observation through fluorescence microscopy.The cytological immunofluorescence observation method is suitable for observing the spindle bodies and the chromosomes of the marine bivalve meiosis device synchronously, has the advantages of simpleand direct operation, high definition and high study efficiency, and is a qualitative, positioning and quantitative cytological immunofluorescence observation method which inosculates forms and functions.

Description

technical field [0001] The invention relates to cytochemistry and cellular immunology, in particular to an immunofluorescent microscopic observation method for seawater bivalve meiosis. Background technique [0002] Marine shellfish is the dominant species of mariculture in China, and occupies a pivotal position in my country's mariculture industry. Utilizing biotechnology to cultivate excellent aquaculture species is one of the key issues to be solved urgently in the development of seawater shellfish aquaculture at present and even in the future. Fertilization is the premise and foundation of individual development and the first step in seed cultivation. In aquaculture production, the quality of fertilization is related to the success or failure of seed cultivation. [0003] Since the 1980s, the chromosome manipulation and cell engineering breeding techniques of seawater economic shellfish have been widely studied, and many advances have been made in the research on the b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/53
Inventor 阙华勇张国范
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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