CN102426126B - Differential staining method for inner cell mass cells and trophoblastic cells of cattle blastulae - Google Patents
Differential staining method for inner cell mass cells and trophoblastic cells of cattle blastulae Download PDFInfo
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- CN102426126B CN102426126B CN 201110251106 CN201110251106A CN102426126B CN 102426126 B CN102426126 B CN 102426126B CN 201110251106 CN201110251106 CN 201110251106 CN 201110251106 A CN201110251106 A CN 201110251106A CN 102426126 B CN102426126 B CN 102426126B
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- 210000004027 cell Anatomy 0.000 title abstract 6
- 241000283690 Bos taurus Species 0.000 title abstract 5
- 210000000625 blastula Anatomy 0.000 title 1
- 238000007447 staining method Methods 0.000 title 1
- 210000000251 trophoblastic cell Anatomy 0.000 title 1
- 238000012744 immunostaining Methods 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 28
- 102000006277 CDX2 Transcription Factor Human genes 0.000 claims abstract description 20
- 108010083123 CDX2 Transcription Factor Proteins 0.000 claims abstract description 20
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 10
- 238000004043 dyeing Methods 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 18
- 239000012895 dilution Substances 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 12
- 238000010926 purge Methods 0.000 claims description 12
- IGAZHQIYONOHQN-UHFFFAOYSA-N Alexa Fluor 555 Chemical compound C=12C=CC(=N)C(S(O)(=O)=O)=C2OC2=C(S(O)(=O)=O)C(N)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C(O)=O IGAZHQIYONOHQN-UHFFFAOYSA-N 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 7
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 7
- 241000283707 Capra Species 0.000 claims description 5
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 239000006059 cover glass Substances 0.000 claims description 3
- 238000010166 immunofluorescence Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 230000012447 hatching Effects 0.000 claims description 2
- 238000010186 staining Methods 0.000 abstract 4
- 210000002993 trophoblast Anatomy 0.000 abstract 3
- 210000002459 blastocyst Anatomy 0.000 abstract 2
- 210000004703 blastocyst inner cell mass Anatomy 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 17
- 229920002451 polyvinyl alcohol Polymers 0.000 description 17
- 210000001161 mammalian embryo Anatomy 0.000 description 5
- 244000144730 Amygdalus persica Species 0.000 description 4
- 235000006040 Prunus persica var persica Nutrition 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一种牛囊胚内细胞团细胞和滋养层细胞的差异染色方法，包括以下步骤：1)利用免疫染色方法，以抗CDX2单克隆抗体作为一抗，将其与特异表达在牛囊胚滋养层细胞的CDX2分子进行免疫结合；2)利用免疫染色方法，以红色荧光标记的能够与抗CDX2单克隆抗体结合的抗体作为二抗，对清洗后的结合有一抗的牛囊胚进行免疫染色；3)在免疫染色完成后，对牛囊胚进行整体的细胞核荧光染色，以形成差异染色。本发明在CDX2蛋白在滋养层细胞内特异性表达，而在内细胞团细胞上不表达的基础上，这样利用基于CDX2分子的红色免疫染色和DAPI细胞核蓝色染色来进行差异染色，准确率为100％，而且图片清晰漂亮。The invention discloses a method for differentially staining bovine blastocyst inner cell mass cells and trophoblast cells, comprising the following steps: 1) using an immunostaining method, using anti-CDX2 monoclonal antibody as the primary antibody, and combining it with the specific expression in bovine The CDX2 molecule of the blastocyst trophoblast cells was immunocombined; 2) using the immunostaining method, the red fluorescently labeled antibody capable of binding to the anti-CDX2 monoclonal antibody was used as the secondary antibody, and the bovine blastocysts combined with the primary antibody after washing were subjected to Immunostaining; 3) After the immunostaining is completed, the bovine blastocyst is subjected to overall nuclear fluorescent staining to form differential staining. In the present invention, on the basis that CDX2 protein is specifically expressed in trophoblast cells but not expressed in inner cell mass cells, differential staining is performed by red immunostaining based on CDX2 molecules and blue staining of DAPI nuclei, and the accuracy is 100%, and the picture is clear and beautiful.
Description
Technical field
The invention belongs to the mammalian embryology field, relate to a kind of differential dyeing method, particularly a kind of ox blastaea inner cell mass cell and trophocyte's differential dyeing method.
Background technology
Inner cell mass cell number in the blastaea and trophocyte's number and the ratio of the two thereof are one of standards of weighing embryo quality.And the dyeing of these two kinds of cells is called differential dyeing in the embodiment blastaea.The principle of traditional differential dyeing method is interior according to the inner cell mass cell, the trophocyte outside, PI with redness processes 20s, dye peripheral trophocyte, dye all cells with blue Hoechst33342 again, after two kinds of pictures merged, peach peripheral cell was the trophocyte, and blue interior confluent monolayer cells is the inner cell mass cell.
This traditional blastaea differential dyeing method is owing to be according to above-mentioned simply inside and outside position principle, so the accuracy rate of differential dyeing is low, and also there is the unsharp problem of picture in traditional differential dyeing.Therefore, how to seek the clear beautiful blastaea differential dyeing method of a kind of accuracy rate height and picture and become current embryology research field urgent problem.
Summary of the invention
The problem that the present invention solves is to provide a kind of ox blastaea inner cell mass cell and trophocyte's differential dyeing method, improve the accuracy of differential dyeing, and the picture that obtains is clear beautiful.
The present invention is achieved through the following technical solutions:
A kind of ox blastaea inner cell mass cell and trophocyte's differential dyeing method may further comprise the steps:
1) utilizes immuning dyeing method, with anti-CDX2 monoclonal antibody as primary antibodie, itself and specifically expressing are carried out immune combination at ox blastaea trophocyte's CDX2 molecule: the ox blastaea is being contained in the immunostaining liquid of primary antibodie, hatching 10～16h in 4 ℃, then cleaning;
2) utilize immuning dyeing method, anti-as two with the antibody that can be combined with anti-CDX2 monoclonal antibody of red fluorescence mark, the ox blastaea that is combined with primary antibodie after cleaning is carried out immunostaining:
Under the lucifuge condition, the ox blastaea in containing the two immunostaining liquid that resist, is hatched 2h in the room temperature lucifuge, then clean;
3) after immunostaining is finished, the ox blastaea is carried out whole nucleus fluorescent dye, to form differential dyeing: the ox blastaea in nucleus fluorescent dye liquid, is hatched 3～5min in the room temperature lucifuge, then clean;
4) the ox blastaea is placed on the microslide, presses with cover glass, then take a picture with fluorescent microscope.
Described primary antibodie is anti-CDX2 mouse monoclonal antibody, and two anti-are Alexa Fluor 555 mark goat anti-mouse IgG; With DAPI the ox blastaea is carried out whole nucleus fluorescent dye.
The described immunostaining liquid that contains primary antibodie is will resist 100～200 times of CDX2 mouse monoclonal antibody dilutions with immunostaining primary antibodie dilution; The described two anti-immunostaining liquid that contain are with 500 times of Alexa Fluor 555 fluorescence labeling goat anti-mouse IgG lucifuges dilutions with immunofluorescence two anti-dilutions.
Described cleaning be with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min.
Described ox blastaea also carries out following pre-service when carrying out immunostaining with primary antibodie:
1) with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min; Described PBS-PVA solution is to contain the PBS solution that volumetric concentration is 0.2%PVA;
2) the ox blastaea is transferred in the immunostaining immobile liquid fixing, in incubated at room 1～2h or 4 ℃ of overnight incubation;
3) will fix rear ox blastaea and transfer to contain in the PBS solution that volumetric concentration is 0.2%Triton X-100 thoroughly and change, hatch 30～45min;
4) with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min;
5) the ox blastaea is transferred in the immunostaining confining liquid sealed, hatch 10～12h in 4 ℃; Again with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min; Finish pre-service.
Compared with prior art, the present invention has following useful technique effect:
The present invention distinguishes ox blastaea trophocyte and inner cell mass cell by seeking a kind of trophocyte's specific expression protein by this albumen being carried out immunostaining.CDX2 albumen is expressed at trophocyte's internal specific, and does not express on the inner cell mass cell.
Therefore, then anti-with the two resistive connections unification of rubescent look fluorescence with the specific antibody dyeing ox blastaea of CDX2 albumen, so the cell of rubescent look fluorescence is the cell of CDX2 protein expression, the expression trophocyte; With DAPI dyeing ox blastaea all cells nuclear, the blastaea all cells look fluorescence that all turns blue, the total cell of expression blastaea.Like this fluorescent dye superimposed after, present pink behind the superimposed blue-fluorescence of red fluorescence, so peach cell is the trophocyte, blue cell is the inner cell mass cell.
Compare with traditional differential dyeing method, the differential dyeing method based on CDX2 dyeing does not have false positive, and the accuracy rate of ox blastaea inner cell mass cell and trophocyte's differential dyeing is 100%, and picture is clear beautiful, is conducive to embry progress.
Description of drawings
Fig. 1 is the microphoto based on the red immunostaining result of CDX2 molecule; The cell of rubescent look fluorescence is the trophocyte;
Fig. 2 is the microphoto of DAPI dyeing ox blastaea all cells nuclear; Cell all presents blue-fluorescence, the total cellular score of expression blastaea;
Fig. 3 is the microphoto of the superimposed differential dyeing of the red immunostaining of CDX2 molecule and DAPI nucleus blue dyeing; Owing to present pink behind the superimposed blue-fluorescence of red fluorescence, so peach cell is the trophocyte, blue cell is the inner cell mass cell.
Embodiment
The invention provides a kind of ox blastaea inner cell mass cell and trophocyte's differential dyeing method, express at trophocyte's internal specific at CDX2 albumen, and on the basis of not expressing on the inner cell mass cell, utilize like this red immunostaining and DAPI nucleus blue dyeing based on the CDX2 molecule to carry out differential dyeing.The present invention is described in further detail below in conjunction with concrete dyeing course and coloration result, and the explanation of the invention is not limited.
A kind of ox blastaea inner cell mass cell and trophocyte's differential dyeing method may further comprise the steps:
Owing to need to carry out immunostaining to the ox blastaea, at first the ox blastaea be carried out following pre-service:
A, with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min; Described PBS-PVA solution is for containing the PBS solution that volumetric concentration is 0.2%PVA (polyvinyl alcohol, polyvinyl alcohol (PVA));
B, the ox blastaea is transferred in the immunostaining immobile liquid (P0098, the green skies, Jiangsu, China) fixing, in incubated at room 1～2h or 4 ℃ of overnight incubation;
C, will fix rear ox blastaea and transfer to contain in the PBS solution that volumetric concentration is 0.2%Triton X-100 thoroughly and change, hatch 30～45min;
D, with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min;
E, the ox blastaea is transferred to sealing in the immunostaining confining liquid (P0102, the green skies), hatch 10～12h in 4 ℃;
F, again with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min; Finish pre-service.
After pre-service is finished, at first carry out the immunostaining of CDX2, carry out again the nucleus bulk dyeing, concrete operations may further comprise the steps:
1) utilize immuning dyeing method, as primary antibodie, itself and specifically expressing carried out immune combination at ox blastaea trophocyte's CDX2 molecule with anti-CDX2 mouse monoclonal antibody (BioGenex, Canada):
To resist CDX2 mouse monoclonal antibody (BioGenex, Canada) with immunostaining primary antibodie dilution (P0103, the green skies) after 100～200 times of dilutions, the ox blastaea is transferred in the immunostaining liquid that contains primary antibodie again, hatch 10～16h in 4 ℃, then clean (purge 3 times, each 5 minutes) with PBS-PVA solution;
Below the operation from step 2) whole lucifuges;
2) utilize immuning dyeing method, the antibody that can be combined with anti-CDX2 mouse monoclonal antibody with the red fluorescence mark is anti-as two, be specially Alexa Fluor 555 mark goat anti-mouse IgG (A0459, the green skies), the ox blastaea that is combined with primary antibodie after cleaning is carried out immunostaining:
Under the lucifuge condition, with two anti-with immunofluorescence two anti-dilutions (P0108, the green skies) lucifuges 500 times of dilutions, again with the ox blastaea in containing two anti-immunostaining liquid, hatch 1～2h in the room temperature lucifuge, then clean with PBS-PVA solution;
3) after immunostaining is finished, the ox blastaea is carried out whole nucleus fluorescent dye, to form differential dyeing:
The ox blastaea is transferred among the DAPI (C1005, the green skies), hatched 3～5min in the room temperature lucifuge, then clean with PBS-PVA solution;
4) the ox blastaea is placed on the microslide, presses with cover glass, then take a picture with fluorescent microscope.
For the result of differential dyeing better is described, specifically describe by reference to the accompanying drawings: Fig. 1 is completing steps 1)～4) afterwards, be the micro-demonstration result who excites under the 555nm exciting light at wavelength, wherein the nucleus of rubescent look fluorescence is trophocyte's nuclear (based on the red immunostaining of CDX2 molecule), expression trophocyte number;
Fig. 2 is completing steps 1)～4) afterwards, be the micro-demonstration result who excites under the 364nm exciting light at wavelength, nucleus all presents blue-fluorescence, the total cellular score of expression blastaea (based on DAPI dyeing ox blastaea all cells nuclear);
Fig. 3 is the microphoto of the differential dyeing of the red immunostaining of CDX2 molecule and DAPI nucleus blue dyeing, also is about to the result that Fig. 1 and Fig. 2 merge; Because each autoluminescence of fluorescence, present pink behind the superimposed blue-fluorescence of red fluorescence, so peach cell is the trophocyte, blue cell is the inner cell mass cell, so just ox blastaea inner cell mass cell and the trophocyte of ox blastaea has been undertaken distinguishing accurately by differential dyeing.
Blastaea inner cell mass cell and trophocyte's cell number and the ratio of the two are important indicators of explanation embryo quality.So the blastaea differential dyeing can be applied to explore the research of embryo's development quality under the various factors condition.For example study the growth whether certain new Embryo Culture environment or certain new embryo medium are conducive to the embryo, can carry out differential dyeing to the blastaea under the specific factor and control group blastaea with the method, whether newer factor has active influence to embryonic development.
Claims (4)
1. an ox blastaea inner cell mass cell and trophocyte's differential dyeing method is characterized in that, may further comprise the steps:
1) utilizes immuning dyeing method, with anti-CDX2 monoclonal antibody as primary antibodie, itself and specifically expressing are carried out immune combination at ox blastaea trophocyte's CDX2 molecule: the ox blastaea is being contained in the immunostaining liquid of primary antibodie, hatching 10～16h in 4 ℃, then cleaning;
Described ox blastaea also carries out following pre-service when carrying out immunostaining with primary antibodie:
A, with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min; Described PBS-PVA solution is to contain the PBS solution that volumetric concentration is 0.2%PVA;
B, the ox blastaea is transferred in the immunostaining immobile liquid fixing, in incubated at room 1～2h or 4 ℃ of overnight incubation;
C, will fix rear ox blastaea and transfer to contain in the PBS solution that volumetric concentration is 0.2%Triton X-100 thoroughly and change, hatch 30～45min;
D, with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min;
E, the ox blastaea transferred in the immunostaining confining liquid seal, hatch 10～12h in 4 ℃; Again with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min; Finish pre-service;
2) utilize immuning dyeing method, anti-as two with the antibody that can be combined with anti-CDX2 monoclonal antibody of red fluorescence mark, the ox blastaea that is combined with primary antibodie after cleaning is carried out immunostaining:
Under the lucifuge condition, the ox blastaea in containing the two immunostaining liquid that resist, is hatched 2h in the room temperature lucifuge, then clean;
3) after immunostaining is finished, the ox blastaea is carried out whole nucleus fluorescent dye, to form differential dyeing: the ox blastaea in nucleus fluorescent dye liquid, is hatched 3～5min in the room temperature lucifuge, then clean;
4) the ox blastaea is placed on the microslide, presses with cover glass, then take a picture with fluorescent microscope.
2. ox blastaea inner cell mass cell as claimed in claim 1 and trophocyte's differential dyeing method is characterized in that, described primary antibodie is anti-CDX2 mouse monoclonal antibody, and two anti-are Alexa Fluor555 mark goat anti-mouse IgG; With DAPI the ox blastaea is carried out whole nucleus fluorescent dye.
3. ox blastaea inner cell mass cell as claimed in claim 1 and trophocyte's differential dyeing method is characterized in that, the described immunostaining liquid that contains primary antibodie is will resist 100～200 times of CDX2 mouse monoclonal antibody dilutions with immunostaining primary antibodie dilution; The described two anti-immunostaining liquid that contain are with 500 times of fluorescently-labeled goat anti-mouse IgG lucifuge dilutions with immunofluorescence two anti-dilutions.
4. ox blastaea inner cell mass cell as claimed in claim 1 and trophocyte's differential dyeing method is characterized in that described step 1), 2), 3) in cleaning be with the ox blastaea in PBS-PVA solution purge repeatedly, each 3～5min.
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| CN102749243B (en) * | 2012-07-16 | 2014-05-14 | 浙江大学舟山海洋研究中心 | Method for staining nucleuses of shrimp embryos |
| CN103245546B (en) * | 2013-05-07 | 2015-07-01 | 东北农业大学 | Simple and rapid pig blastaea differential dyeing method |
| CN103364570B (en) * | 2013-08-02 | 2014-12-17 | 青岛农业大学 | Method for fluorescent double-labeling microscopic observation of marine flounder oosperm microtubules and cell nucleuses |
| CN103397073A (en) * | 2013-08-09 | 2013-11-20 | 广西大学 | Method for simple fluorescence counting of pig blastocysts by utilizing Hoechst33342 |
| CN104166001A (en) * | 2014-07-24 | 2014-11-26 | 中国农业科学院北京畜牧兽医研究所 | Kit for detecting mammal blastula inner cell mass (ICM)/trophectoderm (TE) ratio and cell apoptosis |
| CN104372065A (en) * | 2014-11-20 | 2015-02-25 | 南京优而生物科技发展有限公司 | Method for detecting quality of assisted reproductive technology by mouse embryo array |
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| EP0371839A1 (en) * | 1988-11-30 | 1990-06-06 | L'oreal | Method for the differential colouring of skin cells and their nuclei |
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