CN108254547A - A kind of fluorescence immunoassay group method of body early embryo tissue - Google Patents
A kind of fluorescence immunoassay group method of body early embryo tissue Download PDFInfo
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- CN108254547A CN108254547A CN201810003599.2A CN201810003599A CN108254547A CN 108254547 A CN108254547 A CN 108254547A CN 201810003599 A CN201810003599 A CN 201810003599A CN 108254547 A CN108254547 A CN 108254547A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The invention discloses a kind of fluorescence immunoassay group methods of body early embryo tissue, belong to Animal Biotechnology field.Key step includes embryo's cleaning, fixed, penetrating, removal endogenous peroxydase, closing, primary antibody incubation, secondary antibody incubation and mounting.The technical solution adopted in the present invention has certain specific aim, mainly for body early embryo tissue, and is adjusted and improved on the basis of routine immunization group step.Tissue treatment methods and part are had adjusted using reagent, changes mode of operation, operating time.Operating procedure of the present invention is simple, can effectively solve some technical problems in laboratory inspection analysis, be operated conducive to laboratory practices, compensates for the incomprehensive of existing experimental technique, and actual application value is high.
Description
First, technical field
The present invention relates to a kind of fluorescence immunoassay group methods of body early embryo tissue, belong to Animal Biotechnology field.
2nd, background technology
For mammal origin of life in all-round fertilized eggs, fertilized eggs form different cleavage stage embryos by the spilting of an egg,
It continues to be proliferated and break up, and ultimately forms individual.Early embryonic development is the distinctive growth course of mammal, including from by
For smart ovum to multiple developmental stages such as blastaea, the blastomere before implantation in each phase embryo has very strong plasticity and heterogeneity.Mesh
Before, people are very limited to early embryonic development cognition, and carry out body early embryo research has important meaning to life studies field
Justice.Embryo production in vitro includes zona-free oocytes, lonely male embryo and IVF Embryos must to probe into that early embryonic development provides
The material wanted is a kind of reproduction biotechnology, accelerates Application of Animal Genetic improvement, the important in inhibiting in animal breeding.This
One technology is also an important tool in mammal embryo engineering research, to probe into internal embryonic development mechanism, improvement
Embryonic development quality is had laid a good foundation, be one of internal embryo system effective reflection and Developmental Biology and
An important investigative technique in emerging biometric technology.The research of early embryonic development mechanism be unable to do without immunohistochemistry technique, exempts from
Epidemic disease group technology is applied immunology basic principle, i.e., the principle that antigen is combined with antibody specificity is made by chemical reaction
Labelled antibody chromogenic reagent determines histocyte endoantigen albumen, it is positioned, qualitative and relative quantification is ground
Study carefully, protein positioning directly accurate, qualitative high sensitivity, the technology are widely used, it has also become shape in current biomedicine
State, a powerful of functional metabolism comprehensive study, are one of most important technologies in current life science.This
Application of the technology in embryonic development will provide theoretical reference for researchs such as embryonic development mechanism, embryonic stem cell regulation and control.
3rd, invention content
The purpose of the present invention is to provide a kind of fluorescence immunoassay group methods of body early embryo tissue for technical problem.
Technical solution
1st, a kind of fluorescence immunoassay method of body early embryo tissue, includes the following steps：
1) solution is prepared：4% paraformaldehyde, 0.5%Triton X-100,20% polyvinylpyrrolidone storage solutions
It is dilute with 0.01mol/L phosphate solution of 0.1 μ g/mL4', the 6- diamidino -2-phenylindone with PH ranging from 7.2-7.4
It releases；
2) embryo cleans：Under the microscope, by 2 cell stages of extracorporeal culture, 4 cell stages, 8 cell stages and 16 cell stages
Zona-free oocytes, lonely male embryo or IVF Embryos picked from culture solution, with the addition 6-10%'s preheated at 38.5 DEG C
The 0.01mol/L phosphate buffers of polyvinylpyrrolidone rinse 3 times, each 5min；
3) embryo fixes：It is solid at room temperature with 4% paraformaldehyde of the polyvinylpyrrolidonesolution solution for adding in 6-10%
Determine embryo 1h, then rinsed 3 times with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating, each 5min；
4) it is penetrating：With add in 6-10% polyvinylpyrrolidone 30 min of 0.5%Triton X-100 room temperatures permeabilization,
Then with the phosphate buffer rinsing of the additions polyvinylpyrrolidonesolution solution of preheating 3 times, every time 5 min；
5) endogenous peroxydase is removed：With the 0.01mol/L phosphate for the polyvinylpyrrolidone for adding in 6-10%
Fresh 3%H is configured in buffer solution2O2, room temperature closes 5~10min, with the phosphoric acid of the polyvinylpyrrolidone containing 6-10% of preheating
Salt buffer rinses 3 times, each 5min；
6) it closes：It bleeds and is incubated (with secondary antibody same source) clearly, 37 DEG C of closing 30min；
7) primary antibody is incubated：Confining liquid is gently absorbed, adds and is covered in embryo with the diluted primary antibody of phosphate buffer by antibody explanation
It in tire, places it in wet box, 4 DEG C overnight；
8) secondary antibody is incubated：3 times are rinsed with the phosphate buffer of the polyvinylpyrrolidone containing 6-10% of preheating, every time
5min adds by the diluted fluorescent marker secondary antibody of antibody explanation phosphate buffer, is protected from light in 37 DEG C and is incubated 30min；
9) 4', 6- diamidino -2-phenylindone dye core：Embryo after secondary antibody is incubated uses polyvinylpyrrolidine containing 6-10%
The phosphate buffer of ketone rinses 3 times, each 5min, take 10 μ L, 0.1 μ g/mL4', 6- diamidino -2-phenylindone solution in
On embryo, room temperature 5min；
10) mounting：3 times, each 5min are rinsed with the phosphate buffer of the polyvinylpyrrolidone containing 6-10%, 95%
Glycerine mounting, covered；
11) Fluirescence observation：Fluorescence distribution is observed under Laser Scanning Confocal Microscope.
Advantageous effect
The present invention provides a kind of body early embryo histofluorescence ImmunohistochemistryMethods Methods.Body early embryo histofluorescence of the present invention
ImmunohistochemistryMethods Methods have the following advantages：1) easy to operate, effect is good；2) directly accurate, qualitative high sensitivity is positioned；3) have
There is certain specific aim.The method of the present invention and result are to carry out and early stage lonely female, lonely male or related IVF Embryos biology
It learns research and provides new investigative technique.
Description of the drawings
Fig. 1 is zona-free oocytes growth course；Wherein figure a is 2-4 cell stage blastomeres；Figure b is 4-16 cell stage blastomeres；
Specific embodiment
1st, implement material
The early stage zona-free oocytes of external structure, including before the spilting of an egg, 2 cell stages, 8 cell stages and 16 cell stages.
2nd, implementation
1) solution is prepared：4% paraformaldehyde, 0.5%Triton X-100,20% polyvinylpyrrolidone storage solutions
It is dilute with 0.01mol/L phosphate solution of 0.1 μ g/mL4', the 6- diamidino -2-phenylindone with PH ranging from 7.2-7.4
It releases；
2) embryo cleans：Under the microscope, by 2 cell stages of extracorporeal culture, 4 cell stages, 8 cell stages and 16 cell stages
Zona-free oocytes, lonely male embryo or IVF Embryos picked from culture solution, it is poly- with the addition 8% preheated at 38.5 DEG C
The 0.01mol/L phosphate buffers of vinylpyrrolidone rinse 3 times, each 5min；
3) embryo fixes：It is fixed at room temperature with 4% paraformaldehyde of the polyvinylpyrrolidonesolution solution for adding in 8%
Then embryo 1h is rinsed 3 times, each 5min with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating；
4) it is penetrating：With add in 8% polyvinylpyrrolidone 0.5%Triton X-100 room temperature permeabilization 30min, so
It is rinsed 3 times with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating afterwards, each 5min；
5) endogenous peroxydase is removed：Delayed with the 0.01mol/L phosphate for the polyvinylpyrrolidone for adding in 8%
Fresh 3%H is configured in fliud flushing2O2, room temperature closes 5~10min, with the phosphate buffer containing polyvinylpyrrolidone of preheating
Rinsing 3 times, each 5min；
6) it closes：It bleeds and is incubated (with secondary antibody same source) clearly, 37 DEG C of closing 30min；
7) primary antibody is incubated：Confining liquid is gently absorbed, adds 1:The diluted anti-MT1 polyclonal antibodies of 100 phosphate buffers cover
It is placed in embryo, places it in wet box, 4 DEG C overnight；
8) secondary antibody is incubated：It is rinsed 3 times with the phosphate buffer containing polyvinylpyrrolidone, each 5min adds 1:100
The diluted CY3 of phosphate buffer marks goat-anti rabbit fluorescence secondary antibody, is protected from light in 37 DEG C and is incubated 30min；
9) 4', 6- diamidino -2-phenylindone dye core：Phosphorus of the embryo containing polyvinylpyrrolidone after secondary antibody incubation
Phthalate buffer rinses 3 times, each 5min, takes appropriate 0.1 μ g/mL 4', 6- diamidino -2-phenylindone on embryo, room
Warm 5min；
10) mounting：It is rinsed 3 times, each 5min with the phosphate buffer containing polyvinylpyrrolidone, 95% glycerine envelope
Piece, covered；
11) Fluirescence observation：Fluorescence distribution is observed under Laser Scanning Confocal Microscope.
3rd, treatment effect
Fig. 2 shows that the expression of MT1 albumen changes with embryonic development；When embryo degenerates, expression of receptor gradually subtracts
Weak (figure A, B, C)；And it is mainly expressed (figure D, E, F) in oocyte membrane in the egg mother cell of the non-spilting of an egg；With embryo
The spilting of an egg, expression of receptor gradually increase, and are concentrated mainly on outer rim cell membrane (figure G, H, I)；When embryo continue the spilting of an egg, inside
Begin with expression (the figure J, K, L of receptor；Scheme M, N, O).
Claims (2)
1. a kind of fluorescence immunoassay group method of body early embryo tissue, includes the following steps：
1) solution is prepared：4% paraformaldehyde, 0.5%Triton X-100,20% polyvinylpyrrolidone storage solutions and 0.1 μ
G/mL4', 6- diamidino -2-phenylindone are diluted with the 0.01mol/L phosphate solutions of PH ranging from 7.2-7.4；
2) embryo cleans：Under the microscope, by 2 cell stages of extracorporeal culture, 4 cell stages, 8 cell stages and 16 cell stage early embryos
Tire is picked from culture solution, with the 0.01mol/L phosphate of the polyvinylpyrrolidone of addition 6-10% preheated at 38.5 DEG C
Buffer solution rinses 3 times, each 5min；
3) embryo fixes：Embryo is fixed at room temperature with 4% paraformaldehyde of the polyvinylpyrrolidonesolution solution for adding in 6-10%
Then tire 1h is rinsed 3 times, each 5min with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating；
4) it is penetrating：With add in 6-10% polyvinylpyrrolidone 0.5%Triton X-100 room temperature permeabilization 30min, then
It is rinsed 3 times with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating, each 5min；
5) endogenous peroxydase is removed：With the 0.01mol/L phosphate-buffereds for the polyvinylpyrrolidone for adding in 6-10%
Fresh 3%H is configured in liquid2O2, room temperature closing 5-10min, with the rinsing of the phosphate buffer containing polyvinylpyrrolidone of preheating
3 times, each 5min；
6) it closes：It bleeds and is incubated (with secondary antibody same source) clearly, 37 DEG C of closing 30min；
7) primary antibody is incubated：Confining liquid is gently absorbed, adds and is covered in embryo with the diluted primary antibody of phosphate buffer by antibody explanation,
It places it in wet box, 4 DEG C overnight；
8) secondary antibody is incubated：It is rinsed 3 times, each 5min with the phosphate buffer of the polyvinylpyrrolidone containing 6-10% of preheating,
Add by the diluted fluorescent marker secondary antibody of antibody explanation phosphate buffer, be protected from light in 37 DEG C and be incubated 30min；
9) 4', 6- diamidino -2-phenylindone dye core：The phosphorus of embryo's polyvinylpyrrolidone containing 6-10% after secondary antibody incubation
Phthalate buffer rinses 3 times, each 5min, takes 10 μ L, 0.1 μ g/mL4', 6- diamidino -2-phenylindone solution on embryo,
Room temperature 5min；
10) mounting：It is rinsed 3 times, each 5min with the phosphate buffer of the polyvinylpyrrolidone containing 6-10%, 95% glycerine envelope
Piece, covered；
11) Fluirescence observation：Fluorescence distribution is observed under Laser Scanning Confocal Microscope.
2. according to the method described in claim 1, it is characterized in that：The body early embryo is the zona-free oocytes of in vitro culture, orphan
Male embryo and IVF Embryos.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287325A (en) * | 2022-07-11 | 2022-11-04 | 浙江大学 | Method for evaluating embryonic development potential based on ACLY protein expression positioning |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048470A (en) * | 2012-12-20 | 2013-04-17 | 山西医科大学 | Immunofluorescence histochemistry technology |
| WO2017155869A1 (en) * | 2016-03-07 | 2017-09-14 | X-Zell Inc. | Compositions and methods for identifying rare cells |
-
2018
- 2018-01-03 CN CN201810003599.2A patent/CN108254547A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103048470A (en) * | 2012-12-20 | 2013-04-17 | 山西医科大学 | Immunofluorescence histochemistry technology |
| WO2017155869A1 (en) * | 2016-03-07 | 2017-09-14 | X-Zell Inc. | Compositions and methods for identifying rare cells |
Non-Patent Citations (5)
| Title |
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| JUNJIU HUANG，ET AL.: "Telomere susceptibility to cigarette smoke-induced oxidative damage and chromosomal instability of mouse embryos in vitro", 《FREE RADICAL BIOLOGY & MEDICINE》 * |
| 天津市硅酸盐学会和天津水泥工业设计研究院: "《华北地区硅酸盐学会第八届学术技术交流会论文集》", 30 September 2005, 中国建材工业出版社 * |
| 李颖佳: "小鼠胚胎发育过程中内皮和平滑肌标志物的表达变化", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 董方言: "《现代实用中药新剂型新技术》", 30 April 2001, 人民卫生出版社 * |
| 郭立达和陈鸣丽: "《动物细胞分离培养》", 31 August 2015, 重庆大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287325A (en) * | 2022-07-11 | 2022-11-04 | 浙江大学 | Method for evaluating embryonic development potential based on ACLY protein expression positioning |
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