CN110286019B - A kind of whole embryo fixation method of yolk egg with removed membrane end - Google Patents
A kind of whole embryo fixation method of yolk egg with removed membrane end Download PDFInfo
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- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000012528 membrane Substances 0.000 title claims abstract description 28
- 210000002969 egg yolk Anatomy 0.000 title claims description 9
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 42
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 42
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 40
- 235000013601 eggs Nutrition 0.000 claims abstract description 39
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 18
- 230000004720 fertilization Effects 0.000 claims abstract description 14
- 239000002953 phosphate buffered saline Substances 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims abstract description 5
- 229910019142 PO4 Inorganic materials 0.000 claims abstract 5
- 239000008366 buffered solution Substances 0.000 claims abstract 4
- 239000000834 fixative Substances 0.000 claims abstract 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract 4
- 239000010452 phosphate Substances 0.000 claims abstract 4
- 239000000243 solution Substances 0.000 claims description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- 210000002257 embryonic structure Anatomy 0.000 abstract description 15
- 238000003364 immunohistochemistry Methods 0.000 abstract description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 13
- 102000002322 Egg Proteins Human genes 0.000 description 10
- 108010000912 Egg Proteins Proteins 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 210000004681 ovum Anatomy 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 230000000694 effects Effects 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000007836 KH2PO4 Substances 0.000 description 3
- 241000157468 Reinhardtius hippoglossoides Species 0.000 description 3
- 210000001172 blastoderm Anatomy 0.000 description 3
- 238000002991 immunohistochemical analysis Methods 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 210000001534 vitelline membrane Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000035404 Autolysis Diseases 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 241001391944 Commicarpus scandens Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 210000002824 peroxisome Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- 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
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Abstract
本发明提供一种去膜端黄卵的整胚固定方法，是将端黄卵置于三氯乙酸水溶液中进行室温固定；去掉三氯乙酸水溶液，将端黄卵再用多聚甲醛溶液进行固定；用磷酸缓冲溶液(PBS)置换部分多聚甲醛固定液，4℃保存待用；再极爱你个固定后的受精卵的受精膜去掉，得到完整的胚胎，用磷酸缓冲液或甲醇保存，用于后续的整胚免疫组化或细胞学相关研究。本发明的固定方法固定后的端黄卵，胚胎有韧性，去膜时不易破碎，容易得到完整的胚胎。
The invention provides a method for whole embryo fixation of membrane-removed end-yolk eggs. The end-yolk eggs are placed in an aqueous solution of trichloroacetic acid to fix at room temperature; ; Replace part of the paraformaldehyde fixative with phosphate buffered solution (PBS), and store it at 4°C for later use; then remove the fertilization membrane of the fixed fertilized egg to obtain a complete embryo, which is stored in phosphate buffered saline or methanol. For subsequent whole embryo immunohistochemistry or cytology related studies. The end-yolk eggs fixed by the fixing method of the present invention have tough embryos, are not easily broken when the membrane is removed, and are easy to obtain complete embryos.
Description
The invention relates to a split application, a parent application is filed in 2019 in 26.4. 201910342535X, and the invention is named as 'a whole embryo fixing method for membrane-removed terminal yellow eggs'.
Technical Field
The invention belongs to the technical field of embryo fixation research, and particularly relates to a whole embryo fixation method for a membrane-removed yellow ovum.
Background
Immunohistochemical or immunocytochemical techniques are efficient methods for studying the qualitative, localized and relatively quantitative expression of specific proteins in tissue cells using antigen-antibody specific reactions.
Sample fixation is the primary key link in immunohistochemical analysis. From the perspective of immunohistochemical techniques, the immobilization serves to coagulate intracellular proteins, minimizing or terminating the reaction of exogenous and endogenous enzymes; preventing autolysis of the cells, so as not to allow diffusion of the antigen to the interstitial tissue; maintaining the inherent morphology and structure of the tissue; more importantly, the antigenicity of the tissue or the cell is maintained, so that the antigen is not inactivated, the diffusion phenomenon does not occur, and the background color of the immunohistochemical staining is reduced.
The main purpose of sample fixation is to preserve the material inherent in the cell, coagulate or precipitate the inherent material in the cell or in the tissue fluid, such as nucleus, endoplasmic reticulum, mitochondria, golgi apparatus, lysosome, peroxisome, cytoskeleton, tissue fluid, antigen, etc., so that the cell or tissue substantially maintains the state of the material in life.
However, the terminal yellow eggs are distributed in a polar way, have high yolk content and are concentrated in the plant pole; with less protoplasm and a major focus on the animal pole. For example, when a conventional fixing method such as a 4% paraformaldehyde fixing method is adopted, although the fixing effect on the blastoderm part is good, the fixed blastoderm can be completely peeled off for immunohistochemical analysis, the 4% paraformaldehyde cannot solidify liquid egg yolk substances, and a complete embryo cannot be obtained after fixing, so that the whole embryo immunohistochemical analysis is inconvenient. The acetone and the methanol have a solidifying effect on the liquid yolk, have a good solidifying effect on the fertilized eggs after the fertilized membranes are removed, and can obtain complete embryos. But the solidification effect of fertilized eggs without fertilized membrane removal is poor, and if the fertilized eggs are fixed by acetone and methanol and then membrane removal is carried out, complete embryos cannot be obtained.
In order to facilitate the macromolecular protein antibody to enter the egg cell to be combined with the protein (antigen) to be detected, the fertilization membrane is required to be removed when the immunohistochemistry technology is applied to the egg cell. In order to maintain the cells or tissues substantially in the state of the material in life, they are fixed in real time. Therefore, it is not easy to fix the fertilized membrane of a live egg after removing it, and particularly, the fertilized membrane of a terminal yellow egg having a narrow perivitelline space between the fertilized membrane and the yolk membrane must be fixed and then removed. In view of the fact that the fixed terminal yellow ovum can not obtain the membrane-removed complete embryo by the conventional fixing method, the whole embryo fixing method of the membrane-removed terminal yellow ovum needs to be established, and the whole embryo immunohistochemistry or cytology related research is convenient to carry out.
Disclosure of Invention
Aiming at the defects of the conventional fixing method in the fixation of the terminal yellow ovum, the invention provides the whole embryo fixing method of the membrane-removed terminal yellow ovum, which can solve the whole embryo immunohistochemical or cytology related research needs of the terminal yellow ovum.
The whole embryo fixing method for the membrane-removed yellow eggs provided by the invention comprises the following steps:
1) trichloroacetic acid fixation: fixing the yellow-tipped eggs in trichloroacetic acid aqueous solution at room temperature;
the concentration of the trichloroacetic acid aqueous solution is 4-6%;
the room temperature fixing time is 0.5-2 h;
2) fixing paraformaldehyde: removing the trichloroacetic acid aqueous solution, and fixing the yellow-terminated eggs by using paraformaldehyde solution;
the concentration of the paraformaldehyde solution is 4 percent;
3) replacing the stationary liquid: replacing part of paraformaldehyde fixing solution with Phosphate Buffer Solution (PBS), and storing at 4 deg.C;
the Phosphate Buffer Solution (PBS) has the following components: 128mM NaCl,2mM KCl,8mM NaH2PO4,2mM KH2PO4,pH 7.2。
4) Removing the film: removing the fertilization membrane of the fertilized egg fixed in the step 3) to obtain a complete embryo, and storing the complete embryo by using phosphate buffer solution or methanol for subsequent complete embryo immunohistochemistry or cytology related research.
Further, the fixing method specifically includes the following steps:
1) trichloroacetic acid fixation:
sucking the yellow-tipped eggs into a centrifuge tube, removing the solution, adding 4-6% trichloroacetic acid solution, and fixing for 0.5-2 hours at room temperature;
preferably, the trichloroacetic acid is fixed for 0.5 hour by 6 percent concentration or for 1 hour by 4 percent concentration;
2) fixing paraformaldehyde: removing the trichloroacetic acid solution, adding 4% paraformaldehyde solution into the terminal yellow eggs, and fixing at 4 ℃ overnight or at room temperature for 2-3 hours;
3) replacing the stationary liquid: after fixation of paraformaldehyde, replacing the corresponding 3/4-4/5 volume of paraformaldehyde solution with Phosphate Buffer Solution (PBS), and storing at 4 ℃ for later use;
4) removing the film: removing the fertilization membrane of the fertilized eggs treated in the step 3) to obtain fixed complete embryos.
Compared with the common method for fixing fertilized eggs by 4% paraformaldehyde, the method has the following advantages:
1. the fixing method can solidify yolk substances in the terminal yellow eggs, can obtain complete embryos after the membranes are removed, and is particularly suitable for terminal yellow eggs with higher yolk content in the early stage of fertilization.
2. The perivitelline space between the fertilization membrane and the yolk membrane of the terminal yellow ovum fixed by the fixing method is enlarged, so that the fertilization membrane can be conveniently removed manually, and the method is particularly suitable for terminal yellow ovum with less perivitelline space.
3. The terminal yellow ovum fixed by the fixing method has toughness, is not easy to break when the membrane is removed, and is easy to obtain a complete embryo.
4. The method has wide application range, and the method can be used for fixing the terminal yellow eggs with higher yolk content such as 2-cells, 4-cells, 8-cells and the like before the blastoderm is formed, and can obtain complete embryos after the membrane is removed, thereby facilitating the relevant research of whole embryo immunohistochemistry or cytology.
Drawings
FIG. 1: 4% trichloroacetic acid for 2 hours, 4% paraformaldehyde for overnight fixation at 4 ℃, wherein a is the fixed embryo and b is the decapsulated embryo;
FIG. 2: fixing with 6% trichloroacetic acid for 0.5 hr, and fixing with 4% paraformaldehyde at 4 deg.C overnight, wherein a is fixed embryo and b is deciduated embryo;
FIG. 3: 6% trichloroacetic acid for 1 hour, 4% paraformaldehyde for overnight at 4 ℃. a is the fixed embryo and b is the embryo after stripping.
Detailed Description
The fixing method specifically comprises the following steps:
1) trichloroacetic acid fixation:
sucking the yellow-tipped eggs into a centrifuge tube, removing the solution, adding 4-6% trichloroacetic acid solution, and fixing for 0.5-2 hours at room temperature. The length of the fixing time is related to the use concentration of trichloroacetic acid, the higher the use concentration is, the stronger the protein concentration effect is, the more obvious the increase of perivitelline space is, and the fixing time can be properly shortened; conversely, the lower the concentration used, the weaker the protein-concentrating action, the less marked the increase in perivitelline space, and the longer the fixation time can be suitably. According to the size of the perivitelline space, the shortest fixing time of trichloroacetic acid with the concentration of 6% is 0.5 hour, and the shortest fixing time of trichloroacetic acid with the concentration of 4% is 1 hour.
2) Fixing paraformaldehyde: the trichloroacetic acid solution was decanted, 4% paraformaldehyde solution was added and fixed at 4 ℃ overnight.
3) Replacing the stationary liquid: after fixation with paraformaldehyde, phosphate buffer (PBS component: 128mM NaCl,2mM KCl,8mM NaH) was added2PO4,2mM KH2PO4, pH 7.2) to displace a paraformaldehyde fixing solution of corresponding volume 3/4-4/5 and store at 4 ℃ for later use; the fixing solution is replaced by the phosphate buffer solution without repeated washing, so that the long-term storage of the sample is facilitated, and the dyeing effect is not influenced.
4) Removing the film: and (3) obviously forming perivitelline gaps between the fertilization membranes and the vitelline membranes of the fixed fertilized eggs, removing the fertilization membranes by using a sharp-pointed forceps or a dissecting needle under a dissecting mirror to obtain complete embryos, and storing the complete embryos at 4 ℃ by using Phosphate Buffered Saline (PBS) or freezing and storing 100% methanol at-20 ℃ for subsequent complete embryo immunohistochemistry or cytology related research.
The invention is described in detail below with reference to the figures and examples.
Example 1
Fertilized eggs of the turbot 20min after fertilization are fixed for 2 hours by 4 percent trichloroacetic acid and then fixed overnight by 4 percent paraformaldehyde at 4 ℃. The specific process is as follows:
(1) trichloroacetic acid fixation: the yellow-tipped eggs are sucked into a centrifuge tube by a plastic pipette, after the solution is removed, a 4% trichloroacetic acid solution is added, and the solution is fixed for 2 hours at room temperature.
(2) Fixing paraformaldehyde: the trichloroacetic acid solution was decanted, 4% paraformaldehyde solution was added and fixed at 4 ℃ overnight.
(3) Replacing the stationary liquid: after fixation with paraformaldehyde, phosphate buffer (PBS component: 128mM NaCl,2mM KCl,8mM NaH) was added2PO4,2mM KH2PO4, pH 7.2) was displaced with the corresponding 3/4-4/5 volume of paraformaldehyde fixing solution and stored at 4 ℃ until use.
(4) Removing the film: after fixation, the embryos showed significant perivitelline space (FIG. 1a), and the embryos after membrane removal were stored in phosphate buffer at 4 ℃ until use (FIG. 1 b). As can be seen from fig. 1a and 1 b: the embryo after fixation has less shrinkage and the embryo is complete after membrane removal.
Example 2
Fertilized eggs of the turbot 20min after fertilization are firstly fixed for 0.5 hour by 6 percent trichloroacetic acid and then fixed overnight at 4 ℃ by 4 percent paraformaldehyde. The specific process is as follows:
(1) trichloroacetic acid fixation: the yellow-tipped eggs are sucked into a centrifuge tube by a plastic pipette, 6 percent trichloroacetic acid solution is added after the solution is removed, and the solution is fixed for 0.5 hour at room temperature.
(2) Fixing paraformaldehyde: the trichloroacetic acid solution was decanted, 4% paraformaldehyde solution was added and fixed at 4 ℃ overnight.
(3) Replacing the stationary liquid: after fixation of paraformaldehyde, replacing the corresponding 3/4-4/5 volume of paraformaldehyde fixing solution with Phosphate Buffered Saline (PBS), and storing at 4 ℃ for later use.
(4) Removing the film: the perivitelline space of the fixed embryos is clearly visible (see FIG. 2a), and the embryos after membrane removal are stored in phosphate buffer at 4 ℃ for later use (see FIG. 2 b). As can be seen from fig. 2a and 2 b: the perivitelline space of the fixed embryo is more obvious than that of example 1, and the embryo is complete after membrane removal.
Example 3
Fertilized eggs of the turbot 20min after fertilization are fixed for 1 hour by 6 percent trichloroacetic acid and then fixed overnight by 4 percent paraformaldehyde at 4 ℃. The specific process is as follows:
(1) trichloroacetic acid fixation: the yellow-tipped eggs are sucked into a centrifuge tube by a plastic pipette, 6 percent trichloroacetic acid solution is added after the solution is removed, and the solution is fixed for 1 hour at room temperature.
(2) Fixing paraformaldehyde: the trichloroacetic acid solution was decanted, 4% paraformaldehyde solution was added and fixed at 4 ℃ overnight.
(3) Replacing the stationary liquid: after fixation of paraformaldehyde, replacing the corresponding 3/4-4/5 volume of paraformaldehyde fixing solution with Phosphate Buffered Saline (PBS), and storing at 4 ℃ for later use.
(4) Removing the film: the perivitelline space of the fixed embryos was evident (see FIG. 3a), and the embryos after membrane removal were stored in phosphate buffer at 4 ℃ until use (see FIG. 3 b). As can be seen in fig. 3a and 3 b: the perivitelline space of the fixed embryo is more obvious than that of example 2, and the embryo is complete after membrane removal.
Claims (4)
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Granted publication date: 20211015 |