CN108226470A - A kind of method that embryo's integrality is kept in body early embryo histogenic immunity group - Google Patents

Abstract

The invention discloses a method for maintaining embryo integrity in immunohistochemistry of early embryo tissue, and belongs to the field of animal biotechnology. In this method, polyvinylpyrrolidone solution is added to phosphate buffer, paraformaldehyde, Triton X‑100, and hydrogen peroxide used in early embryo immunohistochemistry to prevent embryos from adhering to the wall and maintain embryo integrity throughout the immunohistochemistry process. Improve the effect of immunohistochemistry. The technical solution adopted in the present invention is mainly aimed at in vitro embryos, and is adjusted on the basis of conventional immunohistochemical steps, and the components of some reagents for tissue processing are adjusted, which effectively improves the effect of immunohistochemistry. The invention aims at the shortcomings of the immunohistochemical technique, expands the scope of application, and has the advantages of simple and convenient operation, low production cost and the like.

Description

A Method for Preserving Embryo Integrity in Immunohistochemistry of Early Embryo Tissue

technical field

The invention relates to a method for maintaining embryo integrity in early embryo tissue immunohistochemistry, and belongs to the field of animal biotechnology.

Background technique

Polyvinylpyrrolidone, referred to as PVP, is a polymer compound produced by the polymerization of N-vinyl-2-pyrrolidone. It is a white hygroscopic powder, odorless or slightly odorous, soluble in water, ethanol, chloroform and most organic solvents, insoluble in ether, and less toxic. Polyvinylpyrrolidone can be synthesized by monomer vinylpyrrolidone through bulk polymerization, solution polymerization and other methods. As a synthetic water-soluble polymer compound, it has the general properties of water-soluble polymer compounds, colloidal protection, film-forming, cohesive, hygroscopic, solubilizing or coagulating effects. Its solution has very low toxicity and is non-physiologically descriptive. It is widely used in medicine, food, cosmetics and other fields.

In vitro embryo culture provides necessary materials for exploring early embryo development. It is a reproductive biotechnology that accelerates the genetic improvement of livestock and poultry, and is of great significance in animal breeding. This technology is also an important tool in the research of mammalian embryo engineering. It has laid a good foundation for exploring the mechanism of embryonic development in vivo and improving the quality of embryonic development. An important research technique in technology. However, during the culture process, the embryos are easy to adhere to the wall, and the capsule wall will be broken when the culture medium is changed to move the embryos, so many embryos will be lost during the whole culture process, and a test sample was constructed. Taking advantage of the water solubility, viscosity and low toxicity of polyvinylpyrrolidone, adding polyvinylpyrrolidone to the embryo culture medium can reduce the adhesion of the embryo and maintain the integrity of the embryo. Based on this, in the embryo immunohistochemical test, the reagents used add a certain concentration The polyvinylpyrrolidone can also reduce the adherence of embryos, maintain the integrity of embryos, and increase the test effect.

The application of this method in embryo immunohistochemistry establishes a feasible test method, which is simple and easy to operate, and the test effect is obvious, which provides a practical basis for embryo engineering research.

Contents of the invention

Technical Problem The object of the present invention is to provide a method for maintaining embryo integrity in immunohistochemistry of early embryo tissue.

Technical solutions

1. A method for maintaining embryo integrity in early embryo tissue immunohistochemistry, comprising the following steps:

1) Solution preparation: 4% paraformaldehyde, 0.5% Triton X-100, hydrogen peroxide, 20% polyvinylpyrrolidone storage solution and 0.1μg/mL 4', 6-diamidino-2-phenylindole are all used in a pH range of 7.2-7.4 was diluted with 0.01mol/L phosphate buffer; and 4% paraformaldehyde, 0.5% Triton X-100, hydrogen peroxide and phosphate buffer were added to the stock solution of polyvinylpyrrolidone for immunohistochemistry. 2%, 4%, 6%, 8%, 10%.

2) Embryo washing: Under a microscope, pick out the parthenogenetic embryos, androgenetic embryos or in vitro fertilized embryos cultivated in vitro at the 2-cell stage, 4-cell stage, 8-cell stage, and 16-cell stage from the culture medium, and wash them at 38.5°C. Rinse with preheated 0.01mol/L phosphate buffer saline with polyvinylpyrrolidone for 3 times, 5min each time;

3) Embryo fixation: fix the embryos with 4% paraformaldehyde added to polyvinylpyrrolidone solution at room temperature for 1 hour, and then rinse with preheated phosphate buffer solution added to polyvinylpyrrolidone solution 3 times, 5 minutes each time;

4) Permeabilization: permeabilize with 0.5% Triton X-100 with polyvinylpyrrolidone at room temperature for 30 min, then rinse with preheated phosphate buffer solution with polyvinylpyrrolidone solution for 3 times, 5 min each time;

5) Remove endogenous peroxidase: prepare fresh 3% H ₂ O ₂ with 0.01mol/L phosphate buffer solution added with polyvinylpyrrolidone, block at room temperature for 5-10min, and use preheated polyvinylpyrrolidone-containing Rinse with phosphate buffer 3 times, 5 min each time;

6) Blocking: Incubate with drops of serum (from the same source as the secondary antibody), and block for 30 min at 37°C;

7) Primary antibody incubation: Remove the blocking solution gently, add the primary antibody diluted with phosphate buffer according to the antibody instruction to cover the embryo, put it in a wet box, and overnight at 4°C;

8) Secondary antibody incubation: wash with preheated phosphate buffer containing polyvinylpyrrolidone for 3 times, each time for 5 minutes, add fluorescently labeled secondary antibody diluted with phosphate buffer according to the antibody instruction, and incubate in the dark at 37°C for 30 min;

9) Nuclei staining with 4',6-diamidino-2-phenylindole: The embryos incubated with the secondary antibody were rinsed 3 times with phosphate buffer containing polyvinylpyrrolidone for 5 minutes each time, and 10 μL of 0.1 μg/ Put mL4',6-diamidino-2-phenylindole solution on the embryo, room temperature for 5min;

10) Mounting: Rinse 3 times with phosphate buffered saline containing polyvinylpyrrolidone, 5 min each time, seal with 95% glycerol, and cover with a coverslip;

11) Fluorescence observation: Observe the fluorescence distribution under a confocal microscope.

Beneficial effect

The present invention provides a method for maintaining embryonic integrity in immunohistochemistry of early embryonic tissue. The invention has the following advantages: 1) easy operation; 2) strong pertinence; 3) improving the immunohistochemical effect of embryonic tissue. The method and results of the invention provide a new research technology for carrying out biological research related to early parthenogenetic, parthenogenetic or in vitro fertilized embryos.

Instructions attached

Figure 1 shows the development process of parthenogenetic embryos; Figure a shows the blastomeres at the 2-4 cell stage; Figure b shows the blastomeres at the 4-16 cell stage.

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified, and the percentages are volume percentages unless otherwise specified.

1. Implementation materials

Early parthenogenetic embryos constructed in vitro, including precleavage, 2-cell embryos, 8-cell embryos and 16-cell embryos.

2. Implementation method

1) Solution preparation: 4% paraformaldehyde, 0.5% Triton X-100, hydrogen peroxide 20% polyvinylpyrrolidone storage solution and 0.1μg/mL 4',6-diamidino-2-phenylindole all use the pH range 7.2-7.4 0.01mol/L phosphate buffer dilution; and immunohistochemistry with 4% paraformaldehyde, 0.5% Triton X-100, hydrogen peroxide and phosphate buffer were added to polyvinylpyrrolidone stock solution, the added concentration 2%, 4%, 6%, 8%, 10% respectively;

2) Embryo washing: Under a microscope, pick out the parthenogenetic embryos, androgenetic embryos or in vitro fertilized embryos cultivated in vitro at the 2-cell stage, 4-cell stage, 8-cell stage, and 16-cell stage from the culture medium, and wash them at 38.5°C. Rinse with preheated 0.01mol/L phosphate buffered saline containing polyvinylpyrrolidone 3 times, 5min each time;

3) Embryo fixation: fix the embryos with 4% paraformaldehyde containing polyvinylpyrrolidone solution at room temperature for 1 hour, then rinse with preheated phosphate buffer solution containing polyvinylpyrrolidone solution 3 times, 5 minutes each time;

4) Permeabilization: Permeabilize with 0.5% Triton X-100 containing polyvinylpyrrolidone at room temperature for 30 min, then rinse with preheated phosphate buffer containing polyvinylpyrrolidone solution 3 times, 5 min each time;

5) Remove endogenous peroxidase: prepare fresh 3% H ₂ O ₂ with 0.01mol/L phosphate buffer containing polyvinylpyrrolidone, block at room temperature for 5-10min, and use preheated polyvinylpyrrolidone-containing Rinse with phosphate buffer 3 times, 5 min each time;

6) Blocking: Incubate with drops of serum (from the same source as the secondary antibody), and block for 30 min at 37°C;

7) Primary antibody incubation: gently aspirate the blocking solution, add 1:100 diluted anti-MT1 polyclonal antibody in phosphate buffer to cover the embryo, put it in a wet box, and overnight at 4°C;

8) Secondary antibody incubation: Rinse 3 times with phosphate buffer containing polyvinylpyrrolidone, 5 min each time, add CY3-labeled goat anti-rabbit fluorescent secondary antibody diluted 1:100 in phosphate buffer, and protect from light at 37°C Incubate for 30 min;

9) Nuclear staining with 4',6-diamidino-2-phenylindole: After incubation with the secondary antibody, wash the embryos with phosphate buffer containing polyvinylpyrrolidone for 3 times, each time for 5 min, take an appropriate amount of 0.1 μg/ Put mL 4',6-diamidino-2-phenylindole on the embryo, room temperature for 5min;

10) Mounting: Rinse 3 times with phosphate buffered saline containing polyvinylpyrrolidone, 5 min each time, seal with 95% glycerol, and cover with a coverslip;

11) Fluorescence observation: Observe the fluorescence distribution under a confocal microscope.

3. Processing effect

Embryos in vitro are easy to adhere to the wall during the culture process, so some embryos cannot guarantee their integrity during some experimental operations, and many embryos are lost. Adding polyvinylpyrrolidone can prevent the embryo from sticking to the wall, which is beneficial to the operation of the embryo in the experiment and maintains the integrity of the embryo. Table 1 shows that during the immunohistochemical test, the integrity of the embryo was greatly affected by the addition of different concentrations of polyvinylpyrrolidone solutions to the reagents used. As the concentration of polyvinylpyrrolidone added increases, the integrity rate of embryos increases gradually, and the integrity of embryos treated with 8% polyvinylpyrrolidone solution is the highest, reaching 95.24%. The completion rate dropped to 72%. It can be seen that in the embryo immunohistochemical test, adding 8% polyvinylpyrrolidone solution, the integrity rate of the embryo is the highest, which increases the test efficiency.

Table 1 Effects of different concentrations of polyvinylpyrrolidone solutions on the integrity of embryos

Polyvinylpyrrolidone solution (%) Number of Embryos (pcs) Number of complete embryos (pieces) Complete rate (%) 2 20 9 45.00 4 twenty two 11 50.00 6 twenty four 18 75.00 8 twenty one 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.