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Immunofluorescence dyeing viewing method for left-eyed flounder zygote microtubule skeleton

A technique for immunofluorescence staining and fertilized eggs, which is applied in the direction of biochemical equipment and methods, microorganisms, microorganisms, etc., to achieve the effects of complete blastodisc structure, sufficient antibody binding, and reduced quenching speed

Inactive Publication Date: 2007-05-02
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the existing immunofluorescence microscopic observation techniques for fertilized egg cytoskeleton of common animals and freshwater fish are not suitable for fertilized eggs of marine flounder and flounder, and a complete set of microtubule skeletons suitable for fertilized eggs of marine flounder and flounder needs to be established. Immunofluorescence staining microscopic observation method

Method used

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  • Immunofluorescence dyeing viewing method for left-eyed flounder zygote microtubule skeleton
  • Immunofluorescence dyeing viewing method for left-eyed flounder zygote microtubule skeleton
  • Immunofluorescence dyeing viewing method for left-eyed flounder zygote microtubule skeleton

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Experimental program
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Effect test

Embodiment 1

[0028] 1) When the flounder fertilized eggs develop to the germinal disc, prepare the FGP-fix fixative solution prepared by the improved PIPES. The method is: first prepare the microtubule polymerization solution-80mM K-PIPES, 5mM EGTA, 100mM MgCl 2 , 400mM NaCl, pH 6.8, stored at 4°C; before fixation, add formaldehyde, glutaraldehyde, and Triton X-100 to the microtubule polymerization solution, and the addition amounts account for 3.7% and 0.25% of the concentration of the microtubule polymerization solution, respectively , 0.5% prepared as a fixative.

[0029] 2) The samples were fixed, and the fertilized eggs were incubated in the fixative at room temperature for 3 hours, then washed twice with PBST buffer, wherein the PBST buffer contained 128mM NaCl, 2mM KCl, 8mMNaH 2 PO 4 , 2mMKH 2 PO 4 , and Tween-20, pH 7.2, the concentration percentage of Tween-20 added is 0.1% of the Tween phosphate buffer solution, and finally stored in PBST at 4°C.

[0030] 3) Under a dissectin...

Embodiment 2

[0036] 1) When turbot fertilized eggs develop to 2-cells, prepare FGP-fix fixative solution prepared by improved PIPES, the method is: first prepare microtubule polymerization solution-90mM K-PIPES, 7mM EGTA, 150mMMgCl 2 , 450mM NaCl, pH 6.9, stored at 4°C; before fixation, add formaldehyde, glutaraldehyde, and Triton X-100 to the microtubule polymerization solution in amounts of 3.8%, 0.4% and 0.75%, prepared as a fixative.

[0037] 2) Fix the sample. After the fertilized eggs are incubated in the fixative at room temperature for 6 hours, wash twice with PBST buffer, wherein the PBST buffer contains 128mM NaCl, 2mM KCl, 8mMNaH 2 PO 4 , 2mMKH 2 PO 4 , and Tween-20, pH 7.3, the concentration percentage of Tween-20 added is 0.2% of the Tween phosphate buffer solution, and finally stored in PBST at 4°C.

[0038] 3) Under a dissecting microscope, the fertilized eggs with a relatively normal development state are selected, and the blastodisc is separated from the whole fertiliz...

Embodiment 3

[0044] 1) Fix the fertilized eggs of turbot at the 4-cell stage, and prepare the FGP-fix fixative solution prepared by improved PIPES. The method is: first prepare the microtubule polymerization solution 100mM K-PIPES, 10mM GTA, 200mM MgCl 2 , 500mM NaCl, pH 7.0, stored at 4°C; before fixation, add formaldehyde, glutaraldehyde, and Triton X-100 to the microtubule polymerization solution in amounts of 4.0%, 0.5% and 1%, prepared as a fixative.

[0045] 2) Fix the fertilized eggs of turbot at the 4-cell stage. After the fertilized eggs are incubated in the fixative at room temperature for 4 hours, they are washed twice with PBST buffer, wherein the PBST buffer contains 128mM NaCl, 2mM KCl, 8mM NaH 2 PO 4 , 2mM KH 2 PO 4 , and Tween-20, pH 7.4, the concentration percentage of Tween-20 added is 0.3% of the Tween phosphate buffer solution, and finally stored in PBST at 4°C.

[0046] 3) Under a dissecting microscope, the fertilized eggs with a relatively normal development state...

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Abstract

An immune fluorescent microscopic observing method of cytula micro-pipe skeleton from bothidae flasfish includes applying fixture solution to fix sample and utilizing dissection needle to separate germ disc from embryon, making hole on sample and using sodium borohydride to eliminate aldehyde group influence on experimental result, carrying out indirect immune fluorescent staining on sample then using fluorescence microscope to observe sample.

Description

technical field [0001] The invention relates to immunofluorescence microscopy technology, in particular to a method for sample fixation, immunofluorescence staining and microscopic observation of the microtubule skeleton of fertilized eggs of sea flounder and flounder. Background technique [0002] Immunofluorescence technology is an effective method to use antigen-antibody specific reaction to study the expression time and location of specific proteins in tissues and cells. The application of immunofluorescence microscopic observation technology in egg cytoskeleton mainly focuses on Drosophila, Xenopus and mouse. The application of marine animal egg cytoskeleton mainly focuses on species such as sea urchins, starfish and prawns. There are few studies on the cytoskeleton of fish eggs by immunofluorescence microscopy, which are only found in freshwater fish such as medaka and zebrafish. The application of microtubule skeleton in seawater fish eggs has not been reported so fa...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/76G01N1/28C12N1/00C12M1/00
Inventor 朱香萍尤锋张培军孙威徐永立
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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