Method of flounder juvenile gonad mRNA in-situ hybridization
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A technology of in situ hybridization and RNA probe, which is applied in the field of mRNA in situ hybridization of juvenile gonads, and can solve the problems of difficult separation, small gonads, and difficult application.
Inactive Publication Date: 2016-06-01
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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The expression pattern and relationship of sex determination and differentiation-related genes can be analyzed by in situ hybridization of gonad mRNA in juvenile flounder, but the gonads of juvenile flounder are small and difficult to separate in the early stage, and it is difficult to directly perform gonad in situ when the gonad tissue is large. hybridization, making the technique difficult to apply
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Embodiment 1
[0029] 1. Sampling and washing.
[0030] The sample is a juvenile fish whose full length is less than 60.0mm and gets its whole abdominal sample; the sample is washed with RNase-free 1×PBS buffer (1×PBS buffer composition is 136.89mMNaCl, 2.67mMKCl, 8.24mMNa 2 HPO 4 ; 1.76mMKH 2 PO 4 , configured by RNase-free water, pH7.4) for rinsing;
[0031] 2. Fixed. After washing, the above samples were placed in RNase-free centrifuge tubes, mixed with 4% PFA at a volume ratio of 1:10, then inverted and mixed, placed flat at 4°C, and fixed for 10 hours. The PFA was obtained by dissolving 4 g of PFA in 100 ml RNase-free 1×PBS buffer.
[0032] 3. Long-term preservation. The above-mentioned fixed-treated samples were rinsed twice with 100% methanol solution, then replaced in 4% PFA solution, and placed at -20°C for long-term storage.
[0033] 4. Dissection. The above-mentioned preserved samples after fixation were removed as much as possible for further processing to obtain in situ ...
Embodiment 2
[0061] 1. Sampling and washing.
[0062] The sample is a gonad sample directly stripped from a juvenile fish with a full length greater than 60.0mm, and the sample is washed with RNase-free 1×PBS buffer (1×PBS buffer composition is 136.89mMNaCl, 2.67mMKCl, 8.24mMNa 2 HPO 4 ; 1.76mMKH 2 PO 4 , configured by RNase-free water, pH7.4) for rinsing;
[0063] 2. Fixed. After washing, the above samples were placed in RNase-free centrifuge tubes, mixed with 4% PFA at a volume ratio of 1:10, then inverted and mixed, placed flat at 4°C, and fixed for 10 hours.
[0064] 3. Long-term preservation. The above-mentioned fixed-treated samples were rinsed twice with 100% methanol solution, then replaced in 4% PFA solution, and placed at -20°C for long-term storage.
[0065] 4. Dissection. The above-mentioned preserved samples after fixation were further processed (removal of the mesangium of the gonad samples, cutting the gonads into a size of about 5 mm) to obtain in situ hybridization ...
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Abstract
The invention relates to juvenile fish gonad mRNA in-situ hybridization technology, in particular to a method of flounder juvenile gonad mRNA in-situ hybridization. The method includes: taking a washed sample, and fixing the washed sample through a PFA solution of 4% in amount concentration; after fixing, using 100% methanol solution for storage of the sample at minus 20 DEG C, and using the fixed and stored sample after being treated as an in-situ hybridization sample; performing rehydration, washing, protease K digestion and fixing on the in-situ hybridization sample, subjecting the in-situ hybridization sample to washing and pre-hybridization, and mixing with hybridization liquid containing a flounder RNA probe after pre-hybridization for hybridization overnight at 50-70 DEG C; washing after hybridization, performing antibody incubation, re-washing, light-avoiding color rendering, color rendering stopping, re-fixing, re-washing, and settling with 20-30%; subjecting a settling sample to OCT embedding, and performing freeze slicing to realize marking of flounder juvenile gonad mRNA in-situ hybridization. By implementing the method, positioning (mRNA level) of flounder related genes in gonad can be clearly described, and breakthrough progress is made.
Description
technical field [0001] The invention relates to an in situ hybridization technique for gonad mRNA of juvenile fish, in particular to a method for in situ hybridization of gonad mRNA of flounder juvenile fish. 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 relat...
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