CN105648065A - Method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization - Google Patents
Method suitable for bastard halibut gonad frozen section mRNA in-situ hybridization Download PDFInfo
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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
The present invention relates to mRNA hybridization in situ technique, specifically a kind of method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization.
Background technology
Paralichthys olivaceus, is commonly called as tooth sheet, and for left-eyed flounder, record belongs to Pleuronectiformes, Paralichthys olivaceus section, Paralichthys, is the important marine fish of the state such as China and Japan and Korea S. Paralichthys olivaceus has milter big feature faster than raun growth, individual, Paralichthys olivaceus progressively becomes marine fish pattern species in Sex determination and differentiation research field, study the mechanism of its Sex determination and differentiation, determine the gene that Sex determination is relevant to differentiation, and based on this, it is implemented sex controll, there is important theory and practice meaning. Expression pattern and the mutual relation of Sex determination and differentiation associated gene can be resolved by Paralichthys olivaceus gonad frozen section mRNA in situ hybridization, but extremely difficult owing to being made directly mRNA whole mount in situ hybridization so that and this technology is difficult to apply. The method hence setting up Paralichthys olivaceus gonad frozen section mRNA in situ hybridization, the elaboration for Paralichthys olivaceus Sex determination and differentiation related law has great importance; Additionally this invention will have certain application prospect in the in situ hybridization that other Fish gonad etc. is organized carries out gene mapping and functional study, it might even be possible to develops coherent detection test kit and carries out industrialization production.
Summary of the invention
A kind of method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization of offer is provided.
For achieving the above object, the technical solution used in the present invention is:
A kind of method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization:
1) it is that 4%PFA solution is fixing and 100% methanol solution is penetrating by the Paralichthys olivaceus gonad amount of sequentially passing through concentration, then preserves for a long time; The sample preserved after fixing, after rehydration, the sedimentation of 20-30% sucrose, carries out OCT embedding and frozen section, 55 DEG C of dry 2-3h, it is thus achieved that frozen section is as in situ hybridization sample;
2) above-mentioned sample through excessive DEPC water washing 3-5min to remove sample sections OCT embedding medium; Washing, protease K digesting, fixing and after after prehybridization, prehybridization, be mixed in 50-70 DEG C with the hybridization solution containing Paralichthys olivaceus rna probe at hybridized overnight; Hybridization process after washing, and through antibody incubation, then wash, lucifuge develops the color, color development stopping, then fixes, mounting, and then realizes the Paralichthys olivaceus gonad related gene expression and localization in mRNA level in-site.
Described fixing after sample after excessive concentrations 100% methanol solution washs again with 100% methanol solution in-20 DEG C long-term preservations.
By in situ hybridization sample through excessive DEPC water washing 3-5min to remove OCT embedding medium; With excessive 1 �� PBST buffer solution cyclic washing without RNase after washing, E.C. 3.4.21.64 (10 �� g/ml) is added in 37 DEG C of digestion process 3-20min after washing, it is that 4%PFA solution fixes 20-60min again in excessive concentrations after digestion process, through excessive 1 �� PBST buffer cyclic washing without RNase after fixing; Iris out sample area with PAP pen after washing, make sample and excessive prehybridization solution in 50-70 DEG C of prehybridization 2-5h.
Described prehybridization solution is 50 (v) % deionized formamide, 25 (v) %20XSSC (raw work, China), 50 �� g/ml heparin, 500 �� g/ml yeast tRNA, the citric acid 460 �� l of 0.1 (v) %Tween-20,1M, adds and is settled to 50ml without RNase water.
The described hybridization solution containing Paralichthys olivaceus rna probe is will to dilute rna probe to final concentration of 1-2ng/ �� l with prehybridization solution, then is placed on 80 DEG C of degeneration 30min, after degeneration, stand-by.
Described probe is rna probe, is build probe carrier with the cDNA of expressing gene in gonad for template, transcribe in vitro synthesis with digoxigenin labeled and one section of strand cRNA molecule being combined with the nucleotide sequence complementary of this gene. Wherein, the gonad development related gene such as nr5a2, nr0b1, cyp19a, dmrt1, foxl2 all can be used to build and synthesize rna probe.
After described prehybridization sample and hybridization solution containing Paralichthys olivaceus rna probe be mixed in 50-70 DEG C at after hybridized overnight at 50-70 DEG C successively prehybridization solution (without heparin and yeast tRNA) short rinse once (5-20min), after rinsing with the prehybridization solution (without heparin and yeast tRNA) that excessive volume ratio is 1:1 and 2 �� SSC mixed liquor washing 5-20min, again with excessive 2 �� SSC washing 10-30min, again with excessive twice washing of 0.2 �� SSC containing 0.1CHAPS, each 15-30min; Last at room temperature with MAB solution washing 5-10min.
Described MAB solution is 100mM maleic acid, 150mMNaCl, pH7.5.
After described hybridization processes washing, sample excessive confining liquid horizontal shaker under room temperature closes 2-4h, then add alkaline phosphatase antibodies 4 DEG C of overnight incubation, wash again, lucifuge develops the color, color development stopping, then fixes, mounting, and then realize the Paralichthys olivaceus gonad related gene expression and localization in mRNA level in-site.
Present invention have the advantage that
The present invention establishes the method for frozen section mRNA in situ hybridization to study related gene expression pattern in gonad in Paralichthys olivaceus, solve the problem such as Gonad Differentiation later stage and period of maturation gonad whole mount in situ hybridization difficulty bigger, direct, it will help the related law of Paralichthys olivaceus Sex determination and differentiation is expanded on further; Additionally this invention will have certain application prospect in the in situ hybridization that other Fish gonad etc. is organized carries out gene mapping and functional study, it might even be possible to develops coherent detection test kit and carries out industrialization production.
Accompanying drawing explanation
The schematic diagram of the Paralichthys olivaceus nr5a2 gene that Fig. 1 provides for embodiment of the present invention frozen section mRNA expressed in situ in gonad.Wherein, nr5a2 expression pattern in development of ovary third stage, short arrow pointed location is follicular cells, and long arrow pointed location is oocyte.
The schematic diagram of the Paralichthys olivaceus foxl2 gene that Fig. 2 provides for embodiment of the present invention frozen section mRNA expressed in situ in ovary. Wherein, foxl2 expression pattern in development of ovary third stage, short arrow pointed location is follicular cells, and long arrow pointed location is oocyte.
Detailed description of the invention
The experimental procedure of the present invention is described in detail in detail below in conjunction with embodiment.
The invention solves the problem that Paralichthys olivaceus gonadal tissue is difficult to be made directly gonad in situ hybridization, it is possible to clearly describe Paralichthys olivaceus related gene expression and localization (mRNA level in-site) in gonad; Additionally this invention will have certain application prospect in the in situ hybridization that other Fish gonad etc. is organized carries out gene mapping and functional study, it might even be possible to develops coherent detection test kit and carries out industrialization production.
Embodiment
1. sampling, washes sample. The Paralichthys olivaceus gonad sample peeled off, rinses with excessive 1 �� PBS without RNase.
2. fixing. Washing later gonad sample and be placed in without in RNase centrifuge tube, the ratio for 1:10 by volume that sample and concentration are 4%PFA mix, after reverse mixing, lies against 4 DEG C, fixes 12h. Described PFA is dissolved in 100ml by 4gPFA and obtains without 1 �� PBS of RNase.
3. preserve for a long time. Being rinsed twice by above-mentioned fixing rear sample excessive concentrations 100% methanol solution, washing is placed in excessive concentration 100% methanol solution, is placed in-20 DEG C long-term preservations.
4. frozen section. By above-mentioned long-term preservation sample through volume ratio 3:2 and 2:3, concentration is 100% methanol and the 1 �� PBST buffer without RNase carries out rehydration respectively and processes 10-30min; Wash 4 times with 100%PBST again, each 5min. After washing, after rehydration, sample, after 1 �� PBS of 30% sucrose settles, carries out OCT embedding, then ice is cut into the section of 14 ��m, be pasted on in situ hybridization anticreep microscope slide, in 55 DEG C of dry 2h, it is thus achieved that frozen section is as in situ hybridization sample after sedimentation;
Described 1 �� PBST buffer components without RNase is: 136.89mMNaCl, 2.67mMKCl, 8.24mMNa2HPO4,1.76mMKH2PO4,0.1 (v) %Tween-20, is configured by without RNase water, PH7.4.
5. washing. By frozen section in situ hybridization sample through DEPC water washing 5min to remove OCT embedding medium; Again with the excessive PBST buffer solution without RNase three times, each 5min after washing.
6. protease K digesting. Frozen section sample E.C. 3.4.21.64 after above-mentioned eluting (10 �� g/ml) is processed 5min in 37 DEG C.
7. fix again. Frozen section sample after above-mentioned digestion is rinsed once with excessive 4%PFA solution, then on room temperature horizontal shaker, fixes 20min again with excessive 4%PFA solution.
8. washing. Rinse once without RNase 1 �� PBST by excessive concentration 100% after fixing, then wash three times without RNase 1 �� PBST by excessive concentration 100%, each 5min.
9. prehybridization. After above-mentioned washing, frozen section sample adds the dosage of 400 �� l prehybridization solutions according to every in situ hybridization anticreep microscope slide, carries out prehybridization in 65 DEG C of hybrid heaters, irises out sample area, prehybridization 4h with PAP pen.
Described prehybridization solution (for 50ml): 50 (v) % deionized formamide, 25 (v) %20XSSC (raw work, China), 50 �� g/ml heparin, 500 �� g/ml yeast tRNA, the citric acid 460 �� l of 0.1 (v) %Tween-20,1M, adds without RNase water to 50ml.
10. hybridization: with prehybridization solution, rna probe being diluted to final concentration 2ng/ �� l, 80 DEG C of degeneration 30min, after above-mentioned hybridization, every in situ hybridization anticreep frost microscope slide, adds the probe that the 130 above-mentioned degeneration of �� l are good, in 65 DEG C of hybridized overnight.
Rna probe is nr5a2 probe or foxl2 probe
The primer sequence building nr5a2 probe carrier is as follows:
F:5��-AAGATCCTGGCGTACCTG-3��
R:5��-ACTCGCTCTTTGTCCTTCA-3��
The primer sequence building foxl2 probe carrier is as follows:
F:5��-GGGAACTACAGGAGACGC-3��
R:5��-CTGTTTTGAAATCACAGGAAATCA-3��
11. post-hybridization washing.
1) hybridization process after at 65 DEG C with excessive 100 (v) % without heparin and yeast tRNA prehybridization solution short rinse once;
2) wash and wash once with 50 (v) % prehybridization solution (without heparin and yeast tRNA) that excessive volume ratio is 1:1 and 50 (v) %2 �� SSC mixed liquor at latter 65 DEG C, 10min;
3) wash once with 2 excessive �� SSC at 65 DEG C, 10min;
4) wash twice containing 0.2 �� SSC containing 0.1CHAPS with excessive at 65 DEG C, each 20min;
5) wash once with excessive MAB solution (100mM maleic acid, 150mMNaCl, pH7.5) under room temperature, 10min.
12. close. Iris out sample area with PAP pen after washing, every microscope slide add 400 �� l confining liquids (1 �� MAB adds 10% sheep blood serum, 2% sealer (Roche, the U.S.), under room temperature close 3h.
13. antibody incubation. Above-mentioned acquisition confining liquid is added primary antibodie (being diluted according to 1:2000 confining liquid), 4 DEG C of overnight incubation.
14. washing.
1) with excessive 100%1 �� PBST buffer short rinse 1 time after antibody incubation;
2) wash 5 times with excessive 1 �� 100%PBST buffer again, each 10min;
3) by sample excesses of basic phosphate buffer (0.1MTris, pH9.5 after above-mentioned washing; 50mMMgCl2; 0.1MNaCl, 0.1%Tween-20) wash 2 times, each 5min;
15. colour developing. With the colour developing of BCIP/NBT (Roche, the U.S.) lucifuge after washing, every 30min dissects Microscopic observation chromogenic reaction;
16. color development stopping. When obvious purple hybridization signal occurs in sample, color development stopping, rinse for several times with excessive 100%PBST buffer after color development stopping, then with the fixing 12h of excessive 4%PFA solution, rinse for several times with excessive 1 �� 100%PBST buffer afterwards.
17. mounting. With 30% glycerol mounting, observe, take pictures, and then realize the Paralichthys olivaceus gonad related gene expression and localization (referring to Fig. 1 or 2) in mRNA level in-site.
Expressed by nr5a2 has at Paralichthys olivaceus ovary as seen from Figure 1, and in follicular cells cell, stronger expression detected.
Expressed by foxl2 has at Paralichthys olivaceus ovary as seen from Figure 2, and in follicular cells cell, stronger expression detected.
Claims (8)
1. the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization, it is characterised in that:
1) it is that 4%PFA solution is fixing and 100% methanol solution is penetrating by the Paralichthys olivaceus gonad amount of sequentially passing through concentration, then preserves for a long time; The sample preserved after fixing, after rehydration, the sedimentation of 20-30% sucrose, carries out OCT embedding and frozen section, 55 DEG C of dry 2-3h, it is thus achieved that frozen section is as in situ hybridization sample;
2) above-mentioned sample through excessive DEPC water washing 3-5min to remove sample sections OCT embedding medium; Washing, protease K digesting, fixing and after after prehybridization, prehybridization, be mixed in 50-70 DEG C with the hybridization solution containing Paralichthys olivaceus rna probe at hybridized overnight; Hybridization process after washing, and through antibody incubation, then wash, lucifuge develops the color, color development stopping, then fixes, mounting, and then realizes the Paralichthys olivaceus gonad related gene expression and localization in mRNA level in-site.
2. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 1, it is characterised in that: described fixing after sample after excessive concentrations 100% methanol solution washs again with 100% methanol solution in-20 DEG C long-term preservations.
3. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 1, it is characterised in that: by situ hybridization sample through excessive DEPC water washing 3-5min to remove OCT embedding medium; With excessive 1 �� PBST buffer solution cyclic washing without RNase after washing, E.C. 3.4.21.64 (10 �� g/ml) is added in 37 DEG C of digestion process 3-20min after washing, it is that 4%PFA solution fixes 20-60min again in excessive concentrations after digestion process, through excessive 1 �� PBST buffer cyclic washing without RNase after fixing; Iris out sample area with PAP pen after washing, make sample and excessive prehybridization solution in 50-70 DEG C of prehybridization 2-5h.
4. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 1, it is characterized in that: described prehybridization solution is 50 (v) % deionized formamide, 25 (v) %20XSSC (raw work, China), 50 �� g/ml heparin, 500 �� g/ml yeast tRNA, 0.1 (v) %Tween-20, the citric acid 460 �� l of 1M, adds and is settled to 50ml without RNase water.
5. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 1, it is characterized in that: the described hybridization solution containing Paralichthys olivaceus rna probe is will to dilute rna probe extremely final concentration of 1-2ng/ �� l with prehybridization solution, it is placed on 80 DEG C of degeneration 30min again, after degeneration, stand-by.
6. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 1, it is characterized in that: after described prehybridization sample and hybridization solution containing Paralichthys olivaceus rna probe be mixed in 50-70 DEG C at after hybridized overnight at 50-70 DEG C with prehybridization solution (without heparin and yeast tRNA) rinsing, 5-20min is washed with the prehybridization solution (without heparin and yeast tRNA) that excessive volume ratio is 1:1 and 2 �� SSC mixed liquor after rinsing, 10-30min is washed again with excessive 2 �� SSC, again with excessive twice washing of 0.2 �� SSC containing 0.1CHAPS, each 15-30min, last at room temperature with MAB solution washing 5-10min.
7. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 6, it is characterised in that: described MAB solution is 100mM maleic acid, 150mMNaCl, pH7.5.
8. by the method being suitable to Paralichthys olivaceus gonad frozen section mRNA in situ hybridization described in claim 1, it is characterized in that: after described hybridization processes washing, sample excessive confining liquid horizontal shaker under room temperature closes 2-4h, then adding alkaline phosphatase antibodies 4 DEG C of overnight incubation, then wash, lucifuge develops the color, color development stopping, fix again, mounting, and then realize the Paralichthys olivaceus gonad related gene expression and localization in mRNA level in-site.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330195A (en) * | 2018-01-25 | 2018-07-27 | 浙江海洋大学 | Labeling method and the application of turbot seminaferous epithelium different development stage reproduction cell and body cell |
| CN109439731A (en) * | 2018-10-23 | 2019-03-08 | 浙江海洋大学 | Genetic marker method suitable for the connection of Sepiella maindroni gap |
| CN110106245A (en) * | 2018-12-03 | 2019-08-09 | 浙江省淡水水产研究所 | A method of suitable for sticking up mouth Culter gonadal tissue mRNA frozen section in situ hybridization |
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| CN101805781A (en) * | 2009-02-13 | 2010-08-18 | 王燕伟 | In situ hybridization optimization and application |
| CN104215471A (en) * | 2014-09-15 | 2014-12-17 | 东华大学 | Preparation method of microRNA in-situ hybridized frozen section |
| CN104878102A (en) * | 2015-05-29 | 2015-09-02 | 中国科学院海洋研究所 | Bastard halibut embryonic-period primordial germ cell tracking and positioning method |
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2016
- 2016-02-05 CN CN201610082004.8A patent/CN105648065A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101805781A (en) * | 2009-02-13 | 2010-08-18 | 王燕伟 | In situ hybridization optimization and application |
| CN104215471A (en) * | 2014-09-15 | 2014-12-17 | 东华大学 | Preparation method of microRNA in-situ hybridized frozen section |
| CN104878102A (en) * | 2015-05-29 | 2015-09-02 | 中国科学院海洋研究所 | Bastard halibut embryonic-period primordial germ cell tracking and positioning method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330195A (en) * | 2018-01-25 | 2018-07-27 | 浙江海洋大学 | Labeling method and the application of turbot seminaferous epithelium different development stage reproduction cell and body cell |
| CN108330195B (en) * | 2018-01-25 | 2021-06-25 | 浙江海洋大学 | Marking method and application of germ cells and somatic cells of turbot spermatogenic epithelium in different development stages |
| CN109439731A (en) * | 2018-10-23 | 2019-03-08 | 浙江海洋大学 | Genetic marker method suitable for the connection of Sepiella maindroni gap |
| CN110106245A (en) * | 2018-12-03 | 2019-08-09 | 浙江省淡水水产研究所 | A method of suitable for sticking up mouth Culter gonadal tissue mRNA frozen section in situ hybridization |
| CN110106245B (en) * | 2018-12-03 | 2022-12-30 | 浙江省淡水水产研究所 | Method suitable for Erythroculter ilishaeformis gonad tissue mRNA frozen section in-situ hybridization |
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