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Method for staining nucleuses of shrimp embryos

A staining method and cell nucleus technology, applied in the preparation of test samples, etc., can solve the problems of interfering with dye penetration, hindering cell nucleus and double-stranded DNA staining effect, etc. Effect

Inactive Publication Date: 2012-10-24
OCEAN RES CENT OF ZHOUSHAN ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that the primary vitelline membrane wrapped outside the shrimp embryo interferes with the penetration of the dye, thereby hindering its staining effect on the nucleus and double-stranded DNA

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Step (1). Shrimp embryos were taken, placed in solution A of 3 times the volume of the embryo, and shaken for 20 minutes at room temperature to obtain the embryo solution after the first treatment;

[0026] Described A solution is the mixed solution of n-heptane and paraformaldehyde solution that the volume ratio is 1:1, wherein the mass content of paraformaldehyde in the paraformaldehyde solution is 4%;

[0027] Step (2). After the embryonic liquid after the first treatment is left to stand for stratification, use a pipette gun to remove the upper liquid phase, and the solid phase obtained is the embryo after the first treatment;

[0028] Step (3). The embryo after the first treatment is re-added with n-heptane of 2 times the volume of the embryo and methanol of 4 times the volume of the embryo, and fully mixed to obtain the embryo solution after the second treatment;

[0029] Step (4). After the embryo solution after the second treatment is left to stand and stratifie...

Embodiment 2

[0037] Step (1). Shrimp embryos were taken, placed in solution A of 5 times the volume of the embryo, and shaken for 30 minutes at room temperature to obtain the embryo solution after the first treatment;

[0038] Described A solution is the mixed solution of n-heptane and paraformaldehyde solution that the volume ratio is 1:1, wherein the mass content of paraformaldehyde in the paraformaldehyde solution is 4%;

[0039] Step (2). After the embryonic liquid after the first treatment is left to stand for stratification, use a pipette gun to remove the upper liquid phase, and the solid phase obtained is the embryo after the first treatment;

[0040] Step (3). The embryo after the first treatment is re-added with n-heptane of 2 times the volume of the embryo and methanol of 4 times the volume of the embryo, and fully mixed to obtain the embryo solution after the second treatment;

[0041] Step (4). After the embryo solution after the second treatment is left to stand and stratifie...

Embodiment 3

[0049] Step (1). Shrimp embryos were taken, placed in solution A of 4 times the volume of the embryo, and shaken for 40 minutes at room temperature to obtain the embryo solution after the first treatment;

[0050] Described A solution is the mixed solution of n-heptane and paraformaldehyde solution that the volume ratio is 1:1, wherein the mass content of paraformaldehyde in the paraformaldehyde solution is 4%;

[0051] Step (2). After the embryonic liquid after the first treatment is left to stand for stratification, use a pipette gun to remove the upper liquid phase, and the solid phase obtained is the embryo after the first treatment;

[0052] Step (3). The embryo after the first treatment is re-added with n-heptane of 2 times the volume of the embryo and methanol of 4 times the volume of the embryo, and fully mixed to obtain the embryo solution after the second treatment;

[0053] Step (4). After the embryo solution after the second treatment is left to stand and stratifie...

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Abstract

The invention discloses a method for staining nucleuses of shrimp embryos. The method includes placing the shrimp embryos in solution A with the volume 3-5 times of that of the embryos at first, and removing upper-layer liquid after standing for layering; adding normal heptane with the volume twice of that of the embryos and methanol with the volume four times of that of the embryos and removing upper-layer solution and middle-layer membranaceous tissues after standing for layering; adding methanol with the volume of 3-5 times of that of the embryos for washing, and repeatedly washing for 3-5 times; adding buffer solution with the volume of 3-5 times of that of the embryos to washing, and repeatedly washing for 3-5 times; staining the nucleuses by soaking the mixture in DAPI (4,6-diamino-2-phenyl indole) solution with the volume of 1-2 times of that of the embryos and the concentration ranging from 5 to 10 micrograms per milliliter in a photophobic manner, and removing liquid after standing for layering; and adding phosphoric acid buffer solution with the volume 3-5 times of that of the embryos for washing, and repeatedly washing for 3-5 times to obtain final embryos. The method has the advantages that an operation method is simple, implementation steps are clear, and a staining effect is clear.

Description

technical field [0001] The invention relates to an embryo cell nucleus staining method, in particular to a shrimp embryo cell nucleus staining method. Background technique [0002] DAPI is 4',6-diamidino-2-phenylindole, which can penetrate the cell membrane and bind to double-stranded DNA in the nucleus to play a labeling role. It is often used for nuclear staining and double-stranded DNA staining in some specific cases . [0003] Shrimp is an arthropod, the body is covered by carapace, and there are many kinds. It has good breeding benefits and industrial prospects. In the field of aquatic seed breeding, it is often necessary to detect and monitor the development process of embryos and observe the real-time progress of embryo development. [0004] At present, the observation of shrimp embryo development is mainly to observe the external morphological changes and development of embryos with a microscope, and there is no suitable detection method to detect the division and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
Inventor 马文明杨卫军钱叶青俞炎琴杨帆杨劲树庄浩瀚
Owner OCEAN RES CENT OF ZHOUSHAN ZHEJIANG UNIV
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