CN108226470A - A kind of method that embryo's integrality is kept in body early embryo histogenic immunity group - Google Patents
A kind of method that embryo's integrality is kept in body early embryo histogenic immunity group Download PDFInfo
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- CN108226470A CN108226470A CN201810021505.4A CN201810021505A CN108226470A CN 108226470 A CN108226470 A CN 108226470A CN 201810021505 A CN201810021505 A CN 201810021505A CN 108226470 A CN108226470 A CN 108226470A
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- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000003037 histogenic effect Effects 0.000 title claims description 8
- 230000036039 immunity Effects 0.000 title claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 34
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 10
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 10
- 238000003364 immunohistochemistry Methods 0.000 claims abstract description 9
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 31
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 30
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 8
- 210000002257 embryonic structure Anatomy 0.000 claims description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- 229920004890 Triton X-100 Polymers 0.000 claims description 6
- 239000013504 Triton X-100 Substances 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 230000003111 delayed effect Effects 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 230000000149 penetrating effect Effects 0.000 claims description 3
- 230000008823 permeabilization Effects 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 8
- 239000012895 dilution Substances 0.000 claims 1
- 238000010790 dilution Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
- 230000001464 adherent effect Effects 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 2
- 238000009472 formulation Methods 0.000 abstract 1
- 230000003053 immunization Effects 0.000 abstract 1
- 238000002649 immunization Methods 0.000 abstract 1
- 238000012309 immunohistochemistry technique Methods 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 229920002113 octoxynol Polymers 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 13
- 241000209094 Oryza Species 0.000 description 4
- 235000007164 Oryza sativa Nutrition 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 235000009566 rice Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 3
- 230000001605 fetal effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000000287 oocyte Anatomy 0.000 description 3
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 210000001109 blastomere Anatomy 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- -1 polyethylene pyrrole Polymers 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 206010011732 Cyst Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000012662 bulk polymerization Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008143 early embryonic development Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Abstract
The invention discloses a kind of methods that embryo's integrality is kept in immunohistochemistry of body early embryo tissue, belong to Animal Biotechnology field.This method is made an addition to using polyvinylpyrrolidonesolution solution in phosphate buffer used in body early embryo immunohistochemistry, paraformaldehyde, Triton X 100, hydrogen peroxide, prevent embryo adherent, embryo's integrality can be kept during entire immunohistochemistry, improves immunohistochemistry effect.The technical solution adopted in the present invention is adjusted on the basis of routine immunization group step mainly for embryo in vitro, has adjusted tissue effecting portion agent formulations, effectively increase immunohistochemistry effect.The present invention is directed to insufficient existing for immunohistochemistry technique, expands application range, with easy to operate, the advantages such as production cost is low.
Description
Technical field
The present invention relates to a kind of methods that embryo's integrality is kept in body early embryo histogenic immunity group, belong to animal life
Object technical field.
Background technology
Polyvinylpyrrolidone abbreviation PVP is the macromolecule chemical combination that polymerization occurs for n-vinyl-2-pyrrolidone
Object.It is that a kind of white has hygroscopic powder, odorless or micro- smelly, water-soluble, ethyl alcohol, chloroform and most organic solvents, no
Ether is dissolved in, toxicity is smaller.Polyvinylpyrrolidone can be using monomers vinylpyrrolidone as raw material, by bulk polymerization, molten
The methods of liquid polymerize synthesizes.As a kind of synthesizing water-solubility high-molecular compound, there is water-soluble high-molecular compound generality
Matter, colloid protective effect, film forming, caking property, hygroscopicity, solubilising or cohesion.Its solution toxicity is very low, is described without physiology
Property.In the fields extensive use such as medicine, food, cosmetics.
Embryo in vitro culture provides necessary material to probe into early embryonic development, is a kind of reproduction biotechnology,
Accelerate Application of Animal Genetic improvement, the important in inhibiting in animal breeding.This technology is also mammal embryo engineering research
In an important tool, for probe into internal embryonic development mechanism, improvement embryonic development quality have laid a good foundation, be
An important research skill in one effective reflection of internal embryo system and Developmental Biology and emerging biometric technology
Art.But in incubation, embryo is easily adherent, can rupture its cyst wall when changing culture solution movement embryo, so entire
Many embryos will be lost in incubation, have built test sample.It is water-soluble, sticky and less toxic using polyvinylpyrrolidone
Property, adds polyvinylpyrrolidone in embryo medium, can reduce that embryo is adherent, keeps the integrality of embryo, based on this,
In the experiment of fetal immune groupization, agents useful for same adds certain density polyvinylpyrrolidone, and it is adherent also to reduce embryo, keeps
Embryo's integrality increases test effect.
Application of this method in fetal immune group establishes feasible test method, easy to operation, experiment effect
Fruit is apparent, and practicable foundation is provided for embryo engineering research.
Invention content
Technical problem keeps embryo complete the purpose of the present invention is to provide one kind in body early embryo histogenic immunity group
The method of property.
Technical solution
1st, a kind of method that embryo's integrality is kept in body early embryo histogenic immunity group, includes the following steps：
1）Solution is prepared：4% paraformaldehyde, 0.5% Triton X-100,20% polyvinylpyrrolidone storage solutions of hydrogen peroxide and
0.1 μ g/mL4', 6- diamidino -2-phenylindone is diluted with the 0.01mol/L phosphate buffers of PH ranging from 7.2-7.4；
And 4% paraformaldehyde of immunohistochemistry, polyethylene pyrrole is added in 0.5% Triton X-100, hydrogen peroxide and phosphate buffer
Pyrrolidone storing liquid, addition concentration is respectively 2%, 4%, 6%, 8%, 10%.
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 are picked from culture solution, with the addition polyvinyl pyrrole preheated at 38.5 DEG C
The 0.01mol/L phosphate buffers of alkanone rinse 3 times, each 5min；
3）Embryo fixes：Embryo 1h is fixed at room temperature with 4% paraformaldehyde for adding in polyvinylpyrrolidonesolution solution, then
It is rinsed 3 times with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating, each 5min；
4）It is penetrating：With 0.5% Triton X-100 room temperatures permeabilization, 30 min for adding in polyvinylpyrrolidone, preheating is then used
The phosphate buffer for adding in polyvinylpyrrolidonesolution solution rinses 3 times, 5 min every time；
5）Remove endogenous peroxydase：It is new with the 0.01mol/L phosphate buffers configuration for adding in polyvinylpyrrolidone
3% fresh H2O2, room temperature closes 5~10min, rinses with phosphate buffer of the preheating containing polyvinylpyrrolidone, often for 3 times
Secondary 5 min；
6）Closing：It bleeds clear（With secondary antibody same source）It is incubated, 37 DEG C of 30 min of closing；
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：Rinse with phosphate buffer of the preheating containing polyvinylpyrrolidone, every time 5 min for 3 times, add by
The diluted fluorescent marker secondary antibody of antibody explanation phosphate buffer is protected from light in 37 DEG C and is incubated 30 min；
9）4', 6- diamidino -2-phenylindone dye core：Embryo after secondary antibody is incubated is delayed with the phosphate containing polyvinylpyrrolidone
Fliud flushing rinses 3 times, every time 5 min, 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 with the phosphate buffer containing polyvinylpyrrolidone, 5 min, 95% glycerine mounting cover every time
Upper coverslip；
11）Fluirescence observation：Fluorescence distribution is observed under Laser Scanning Confocal Microscope.
Advantageous effect
The present invention provides a kind of methods that embryo's integrality is kept in body early embryo histogenic immunity group.The present invention have with
Lower advantage：1）It is easy to operate；2）It is with strong points；3）Improve embryonic tissue immunohistochemistry effect.The method of the present invention and result is open
It opens up and provides new investigative technique with early stage lonely female, lonely male or related IVF Embryos biological study.
Figure of description
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
Method therefor is conventional method unless otherwise instructed in following embodiments, and the percentage composition is unless otherwise instructed
It is volumn concentration.
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 of hydrogen peroxide and
0.01mol/L phosphate buffer of 0.1 μ g/mL 4', the 6- diamidino -2-phenylindone with PH ranging from 7.2-7.4 is dilute
It releases；And 4% paraformaldehyde of immunohistochemistry, poly- second is added in 0.5% Triton X-100, hydrogen peroxide and phosphate buffer
Alkene pyrrolidone storing liquid, addition concentration is respectively 2%, 4%, 6%, 8%, 10%；
2）Embryo cleans：Under the microscope, it is the orphan of 2 cell stages of extracorporeal culture, 4 cell stages, 8 cell stages and 16 cell stages is female
Embryo, lonely male embryo or IVF Embryos pick from culture solution, with what is preheated at 38.5 DEG C containing polyvinylpyrrolidone
0.01mol/L phosphate buffers rinse 3 times, each 5min；
3）Embryo fixes：Embryo 1h, Ran Houyong are fixed at room temperature with 4% paraformaldehyde containing polyvinylpyrrolidonesolution solution
The phosphate buffer containing polyvinylpyrrolidonesolution solution of preheating rinses 3 times, each 5min；
4）It is penetrating：With 0.5% Triton X-100 room temperatures permeabilization, 30 min containing polyvinylpyrrolidone, then being contained with what is preheated
The phosphate buffer of polyvinylpyrrolidonesolution solution rinses 3 times, every time 5 min；
5）Remove endogenous peroxydase：It is fresh with the 0.01mol/L phosphate buffers configuration containing polyvinylpyrrolidone
3% H2O2, room temperature closes 5~10min, rinses with phosphate buffer of the preheating containing polyvinylpyrrolidone, every time for 3 times
5 min；
6）Closing：It bleeds clear（With secondary antibody same source）It is incubated, 37 DEG C of 30 min of closing；
7）Primary antibody is incubated：Confining liquid is gently absorbed, adds 1:The diluted anti-MT1 polyclonal antibodies of 100 phosphate buffers are covered in
It in embryo, places it in wet box, 4 DEG C overnight；
8）Secondary antibody is incubated：With containing polyvinylpyrrolidone phosphate buffer rinse 3 times, 5 min, adds 1 every time:100 phosphorus
The diluted CY3 of phthalate buffer marks goat-anti rabbit fluorescence secondary antibody, is protected from light in 37 DEG C and is incubated 30 min；
9）4', 6- diamidino -2-phenylindone dye core：Embryo after secondary antibody is incubated is delayed with the phosphate containing polyvinylpyrrolidone
Fliud flushing rinses 3 times, every time 5 min, takes appropriate 0.1 μ g/mL 4', 6- diamidino -2-phenylindone on embryo, room temperature 5min；
10）Mounting：It is rinsed 3 times with the phosphate buffer containing polyvinylpyrrolidone, 5 min, 95% glycerine mounting cover every time
Upper coverslip；
11）Fluirescence observation：Fluorescence distribution is observed under Laser Scanning Confocal Microscope.
3rd, treatment effect
Embryo in vitro is easily adherent in incubation, causes some embryos that can not ensure that its is complete during some test operations
Whole property, and lose many embryos.Addition polyvinylpyrrolidone embryo can be prevented adherent, conducive in experiment to the behaviour of embryo
Make, keep embryo's integrality.Table 1 is shown, during immunohistochemical assay, various concentration polyethylene pyrrole is added in agents useful for same
Pyrrolidone solution, embryo's integrality are greatly affected.Increase with the polyvinylpyrrolidoneconcentration concentration of addition, embryo's percentage of head rice
It gradually increases, with 8% polyvinylpyrrolidonesolution solution, treated that embryo's integrality is highest, can reach 95.24%；But work as and add
After adding 10% polyvinylpyrrolidonesolution solution, embryo's percentage of head rice drops to 72%.It can be seen that in the experiment of fetal immune groupization, add
Add 8% polyvinylpyrrolidonesolution solution, the percentage of head rice of embryo is highest, increases test efficiency.
Influence of the 1 various concentration polyvinylpyrrolidonesolution solution of table to embryo's integrality
| Polyvinylpyrrolidonesolution solution（%） | Embryo number（It is a） | Complete embryo number（It is a） | Percentage of head rice（%） |
| 2 | 20 | 9 | 45.00 |
| 4 | 22 | 11 | 50.00 |
| 6 | 24 | 18 | 75.00 |
| 8 | 21 | 20 | 95.24 |
| 10 | 25 | 18 | 72.00 |
Claims (2)
1. a kind of method that embryo's integrality is kept in body early embryo histogenic immunity group, includes the following steps：
1) solution is prepared：4% paraformaldehyde, 0.5%Triton X-100,20% polyvinylpyrrolidone storage solutions of hydrogen peroxide
It is dilute with 0.01mol/L phosphate buffer of 0.1 μ g/mL 4', the 6- diamidino -2-phenylindone with PH ranging from 7.2-7.4
It releases, and 4% paraformaldehyde of immunohistochemistry, is added in 0.5%Triton X-100, hydrogen peroxide and phosphate buffer poly-
Vinylpyrrolidone storing liquid, addition concentration is respectively 2%, 4%, 6%, 8%, 10%；
2) embryo cleans：Under the microscope, it is the orphan of 2 cell stages of extracorporeal culture, 4 cell stages, 8 cell stages and 16 cell stages is female
Embryo, lonely male embryo or IVF Embryos are picked from culture solution, with the addition polyvinylpyrrolidone preheated at 38.5 DEG C
0.01mol/L phosphate buffers rinse 3 times, each 5min；
3) embryo fixes：Embryo 1h is fixed at room temperature with 4% paraformaldehyde for adding in polyvinylpyrrolidonesolution solution, then
It is rinsed 3 times with the phosphate buffer of the addition polyvinylpyrrolidonesolution solution of preheating, each 5min；
4) it is penetrating：With the 0.5%Triton X-100 room temperature permeabilization 30min for adding in polyvinylpyrrolidone, preheating is then used
The phosphate buffer for adding in polyvinylpyrrolidonesolution solution rinses 3 times, each 5min；
5) endogenous peroxydase is removed：It is new with the 0.01mol/L phosphate buffers configuration for adding in polyvinylpyrrolidone
Fresh 3%H2O2, room temperature closing 5-10min rinses, every time for 3 times with phosphate buffer of the preheating containing polyvinylpyrrolidone
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, added by anti-with the phosphate buffer containing polyvinylpyrrolidone of preheating
The diluted fluorescent marker secondary antibody of body 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 is delayed with the phosphate containing polyvinylpyrrolidone
Fliud flushing rinses 3 times, each 5min, takes 10 μ L, 0.1 μ g/mL 4', 6- diamidino -2-phenylindone solution on embryo, room temperature
5min；
10) mounting：It is rinsed 3 times, each 5min with the phosphate buffer containing polyvinylpyrrolidone, 95% glycerine mounting, lid
Upper coverslip；
11) Fluirescence observation：Fluorescence distribution is observed under Laser Scanning Confocal Microscope.
2. a kind of method that embryo's integrality is kept in body early embryo histogenic immunity group according to claim 1,
It is characterized in that：The 0.01mol/L phosphate buffers dilution that the polyvinylpyrrolidonesolution solution is PH ranging from 7.2-7.4 is molten
Liquid.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110286019A (en) * | 2019-04-26 | 2019-09-27 | 青岛农业大学 | A kind of whole embryo fixing means of striping anisolecithal egg |
| CN111190001A (en) * | 2020-01-10 | 2020-05-22 | 苏州睿瀛生物技术有限公司 | Special diluent for immunohistochemical antibody and preparation method thereof |
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| CN111190001B (en) * | 2020-01-10 | 2024-03-26 | 苏州睿瀛生物技术有限公司 | Special diluent for immunohistochemical antibody and preparation method thereof |
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