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Method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization

A technology of frozen section and in situ hybridization, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., and can solve difficult and difficult-to-apply problems

Inactive Publication Date: 2016-06-08
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

The expression pattern and relationship of sex determination and differentiation-related genes can be analyzed by mRNA in situ hybridization of frozen sections of flounder gonads, but it is very difficult to directly perform mRNA in situ hybridization, making this technique difficult to apply

Method used

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  • Method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization
  • Method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization
  • Method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization

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Embodiment

[0023] 1. Sampling and washing. Stripped flounder gonad samples were rinsed with excess RNase-free 1×PBS buffer.

[0024] 2. Fixed. The washed gonad samples were placed in RNase-free centrifuge tubes. The samples were mixed with 4% PFA at a volume ratio of 1:10. After inversion and mixing, they were placed flat at 4°C and fixed for 12 hours. The PFA was obtained by dissolving 4 g of PFA in 100 ml RNase-free 1×PBS buffer.

[0025] 3. Long-term storage. The above-mentioned fixed samples were rinsed twice with an excess concentration of 100% methanol solution, placed in an excess concentration of 100% methanol solution after washing, and stored at -20° C. for a long time.

[0026] 4. Frozen section. The above-mentioned long-term storage samples were rehydrated for 10-30 minutes with the volume ratio of 3:2 and 2:3, the concentration of 100% methanol and RNase-free 1×PBST buffer respectively; then washed 4 times with 100% PBST, 5 minutes each time. After washing and rehydrat...

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Abstract

The invention relates to an mRNA in-situ hybridization technology, in particular to a method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization. The method includes the steps that bastard halibut gonad is immobilized with an excess PFA solution with the concentration of 4% and permeated with an excess methanol solution with the concentration of 100% in sequence and then is preserved for a long term; the sample which is immobilized and then preserved is subjected to rehydration and 20-30% saccharose sedimentation, then is subjected to OCT embedding and frozen section preparing and is dried for 2-3 h at 55 DEG C, and a frozen section is obtained to serve as an in-situ hybridization sample; the sample is washed for 3-5 min with excess DEPC water to remove an OCT embedding agent in the sample frozen section; the sample frozen section is subjected to washing, proteinase K digestion and immobilization, then is subjected to pre-hybridization and then is mixed with a hybridization solution containing a bastard halibut RNA probe at 50-70 DEG C for overnight hybridization; after hybridization treatment, washing, antibody incubation, rewashing, light-proof color development, termination color development, re-immobilization and section encapsulation are carried out, and thus expression localization of bastard halibut gonad related genes at the mRNA level is achieved. By means of the method, the problem that direct gonad in-situ hybridization of bastard halibut gonadal tissue is difficult is solved, and expression localization of bastard halibut related genes in gonad at the mRNA level can be clearly described.

Description

technical field [0001] The invention relates to mRNA in situ hybridization technology, in particular to a method suitable for mRNA in situ hybridization of frozen sections of flounder gonads. Background technique [0002] Flounder, commonly known as tooth slices, is a flounder fish, which belongs to the order Pleurotus, family Flounder, and genus Flounder. It is an important marine cultured fish in my country, Japan, South Korea and other countries. Flounder has the characteristics that males grow faster than females and are larger in size. Flounder has gradually become a model species of seawater economic fish in the field of sex determination and differentiation research. The mechanism of sex determination and differentiation is studied to determine the relationship between sex determination and differentiation. gene, and based on this, it has important theoretical and practical significance to implement sex control on it. The expression pattern and relationship of sex de...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2565/518
Inventor 焦爽范兆飞谭训刚尤锋
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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