CN106226133B - The preparation method of novel C OCs composite construction tissue comprising multiple complexs - Google Patents
The preparation method of novel C OCs composite construction tissue comprising multiple complexs Download PDFInfo
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- CN106226133B CN106226133B CN201610515409.6A CN201610515409A CN106226133B CN 106226133 B CN106226133 B CN 106226133B CN 201610515409 A CN201610515409 A CN 201610515409A CN 106226133 B CN106226133 B CN 106226133B
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- ocs
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- 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
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- 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
- G01N1/36—Embedding or analogous mounting of samples
Abstract
The preparation method of the invention discloses a kind of novel C OCs composite construction tissue comprising multiple complexs obtains cumulus oocytes complesxes using suction or post-mortem method from folliculus ovarii；Using cumulus oocytes complesxes as basic material, by the way of paraffin embedding, cumulus oocytes complesxes are drawn into EP pipe, are impregnated with soft wax and hard wax, injection embedding case is sucked out, is embedded.The present invention constructs a kind of novel C OCs composite construction tissue comprising multiple complexs in the way of paraffin embedding；Prior paraffin histotomy technology is creatively used for cell detection, improve traditional expression process, simplify many and diverse dehydration procedure, significantly shorten detection time, fixed effect is improved, efficiently avoids being not easy to switch to the problem of ovarian follicle is easily lost with egg mother cell in ovarian sections technology；Understand to clear and definite the albumen coordinate expression situation in cumulus cell and egg mother cell.
Description
Technical field
The invention belongs to immunocytochemistries and immunohistochemistry technology field, more particularly to a kind of includes multiple compound
The preparation method of the novel C OCs composite construction tissue of body.
Background technique
Mammal antral follicles are a kind of specific stage of development ovarian follicles occurred after the animals into heat phase.This period ovarian follicle
Interior, egg mother cell is outer by multilayer cumulus cell, only thin with part cumulus granulosa cells and the wall particle that is attached on theca interna
Cell phase connects, and is protruded from the liquor folliculi in antrum folliculi with the specific form of cumulus oocytes complesxes (COCs).With ovarian follicle
Continued development and advantage, graaffian follicle rupture, COCs is discharged into fallopian tubal.This composite construction enters after fallopian tubal, meeting
It rests on ampulla of uterine tube and waits fertilization.This structure and functional characteristics characteristic based on COCs, will in the antral follicles stage
COCs takes out out of ovarian follicle, in suitable vitro, can successfully cultivate COCs to the fertilizable stage of ripeness.Herein
In structure, cumulus cell carries out nutrition transmission and information interchange with egg mother cell in a manner of gap connection etc..Connection between the two
Structure and the information interchange carried out on this basis and function interaction are the basic guarantees of oocyte maturation.So protecting
On the basis of staying COCs structural integrity, certain agnoprotein or certain protein ligands and receptor are inquired into egg mother cell and cumulus cell
On coordinate expression situation disclose ovarian follicle for understanding its possible autocrine or paracrine mode in this structure in depth
Development and oocyte maturation mechanism, there is important Methodological Significance.The qualitative detection specific protein in cell and tissue at present
The routine techniques of expression mainly includes immunocytochemistry and two class technology of immunohistochemistry.But above-mentioned technology exists
Detect mammal ovarian cumulus-oocyte complex (COCs) in protein expression when there are notable defects, be now described below: 1.
Immunocytochemical technique: this is a kind of technology of specific protein expression of detection on a cellular level.COCs is one kind by multilayer
The composite construction of cumulus cell and one layer of homogeneous clear band coating egg mother cell, under conventional laboratory conditions, primary antibody and tape label
Group secondary antibody hardly enters inside the deep layer ovarian cumulus or egg mother cell of the composite construction, causes in cumulus cell and egg mother cell
Antigenic mark to be checked is difficult；2. immunohistochemistry technology: this technology handles fixed group in a manner of paraffin embedding or frost
It knits block, after slice, utilizes specific protein in each cell of immunological technique tagged tissue.Ovarian follicle on ovary can be divided into roughly ovum before chamber
Bubble and antral follicles two major classes.COC only passes through part cumulus granulosa cells and is connected with ovarian follicle wall in antral follicles, and with
The continuous development of ovarian follicle, the connection of connected component become further loose.So when detecting protein expression in COCs using the technology
There are shortcomings, such as: ovarian follicle is not easy to switch to；Switching to ovarian follicle, still COCs or egg mother cell are easily lost；Workload is huge；
Due to, there is also other cells (outer histocyte of wall granular cell, thecacells, ovarian follicle etc.), causing purpose in ovary tissue
Protein urine is indefinite etc. on cell (cumulus cell and egg mother cell).
There are primary antibodies and tape label group for the technology of qualitative detection specific protein expression in cell and tissue at present
Secondary antibody hardly enters inside the deep layer ovarian cumulus or egg mother cell of the composite construction, cause cumulus cell with it is to be checked in egg mother cell
Antigenic mark is difficult, and ovarian follicle is not easy to switch to；Ovarian follicle is switched to, COCs or egg mother cell are easily lost, and workload is huge, and purpose is thin
The upper protein urine of born of the same parents is indefinite.
Summary of the invention
The preparation side of the purpose of the present invention is to provide a kind of novel C OCs composite construction tissue comprising multiple complexs
Method, it is intended to solve at present that there are primary antibodies and tape label base for the technology of qualitative detection specific protein expression in cell and tissue
Group's secondary antibody hardly enters inside the deep layer ovarian cumulus or egg mother cell of the composite construction, cause in cumulus cell and egg mother cell to
It is difficult to examine antigenic mark, ovarian follicle is not easy to switch to；Ovarian follicle is switched to, COCs or egg mother cell are easily lost, and workload is huge, purpose
The indefinite problem of protein urine on cell.
The invention is realized in this way a kind of preparation side of the novel C OCs composite construction tissue comprising multiple complexs
Method, the preparation method of the novel C OCs composite construction tissue comprising multiple complexs is using suction or post-mortem method, from ovum
Cumulus oocytes complesxes are obtained in nest ovarian follicle；Using cumulus oocytes complesxes as basic material, using paraffin embedding
Mode, cumulus oocytes complesxes are drawn into EP pipe, through fixation, washing, dehydration, transparent with waxdip, into
Row embedding；Construct a kind of novel cumulus oocytes complesxes composite construction tissue comprising multiple complexs.
Further, the preparation method of the novel C OCs composite construction tissue comprising multiple complexs specifically includes following
Step:
Step 1 obtains cumulus oocytes complesxes using suction or post-mortem method from folliculus ovarii；
Cumulus oocytes complesxes are placed in paraformaldehyde solution fixed by step 2；
Cumulus oocytes complesxes are incubated for by step 3 in 50% alcohol and dehydrated alcohol；
Step 4 is transferred to diformazan by cumulus oocytes complesxes after solution (dimethylbenzene+dehydrated alcohol 1:1) transition
It is transparent in benzene liquid；
Cumulus oocytes complesxes are drawn into 1.5mlEP pipe by step 5, in dewaxing case, with soft wax and hard wax
It immerses, is advisable so that whole complexs can be covered；The embedding case of injection 1CM square is sucked out, is embedded；
Embedded wax stone is fixed on slicer, thinly slices, be attached on glass slide, puts in insulating box by step 6
Drying；
Step 7 is dewaxed with dimethylbenzene；100% ethyl alcohol is incubated for；
Step 8 is added dropwise 5% secondary antibody source and closes serum, and room temperature is closed 30 minutes in wet box；4 DEG C of refrigerators in primary antibody wet box
It is incubated overnight；Secondary antibody is after magazine is incubated at room temperature 1h, rinsing；
Step 9 is observed with fluorescence microscope or confocal fluorescent microscopic after mounting.
Cumulus oocytes complesxes are placed in 4% paraformaldehyde solution by the step 2 fixes 2 hours.
Cumulus oocytes complesxes are respectively incubated for 20 minutes by the step 3 in 50% alcohol and dehydrated alcohol.
Cumulus oocytes complesxes after solution (dimethylbenzene+dehydrated alcohol 1:1) transition, are transferred to by the step 4
Transparent 10 minutes in 100% dimethylbenzene liquid.
Cumulus oocytes complesxes are drawn into 1.5mlEP pipe by the step 5, and in 60 DEG C of dewaxing casees, use is soft
Wax and hard wax immerse each 30 minutes；The embedding case of injection 1CM square is sucked out, is embedded.
Embedded wax stone is fixed on slicer by the step 6, is cut into a thickness of 5 μm of thin slices.It plates, is attached to load
On slide, puts in 45 DEG C of insulating boxs and dry.
The step 7 is dewaxed 10 minutes with dimethylbenzene；100% ethyl alcohol is incubated for 10 minutes.
Include the novel C OCs composite construction comprising multiple complexs another object of the present invention is to provide a kind of
The frozen section manufacture craft of the preparation method preparation novel C OCs composite construction tissue of tissue.
The present invention has collected multiple cumulus oocytes complesxes for picking up from antral follicles, through fixation, washing, dehydration, thoroughly
After the bright processing with waxdip, paraffin is perfused, is embedded.The spy that multiple cell conjugates can be assembled using paraffin
Point artificially constructs a kind of " novel C OCs composite construction tissue " comprising multiple complexs, creatively by prior paraffin group
Microtomy is knitted for cell detection, simplifies the techniqueflow of traditional immunohistochemical staining；Eliminate routine immunization
Penetrating step in Cytochemical Technique after being sliced to " group COCs ", cell membrane, matter and nucleus can directly be exposed
In dye liquor environment；Many and diverse dehydration procedure is simplified, tradition is organized in microtomy from 50%-60%-70%-80%-
The dewatering process flow of 90% alcohol-dehydrated alcohol, is reduced to 50% alcohol and two step of dehydrated alcohol is dehydrated；When significantly shortening detection
Between, detection technique is organized compared to tradition, the set time was foreshortened to 2 hours etc. by 4 days；Improve fixed effect, animal groups
It knits block ingredient and structure is complicated, in contrast, the only female two class cells of ovarian cumulus and ovum, structure letter in the composite construction tissue of building
It is single, definite ingredients, therefore it is fixed it is time-consuming less, repeatability is high, effect is good；At " the artificial COCs composite construction tissue " of building, only
Ovarian cumulus and the female two kinds of cells of ovum, connection structure keep complete, can with clear and definite understand the albumen coordinate expression in the structure
Situation；It efficiently avoids being not easy to switch to the problem of ovarian follicle is easily lost with egg mother cell in ovarian sections technology；It can also incite somebody to action
" the COCs composite construction tissue " that the present invention constructs makes for frozen section.
Detailed description of the invention
Fig. 1 is the preparation method of the novel C OCs composite construction tissue provided in an embodiment of the present invention comprising multiple complexs
Flow chart.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
The present invention is using cumulus oocytes complesxes as basic material, in a manner of paraffin embedding, artificially constructs a kind of packet
" novel C OCs composite construction tissue " containing multiple complexs, improves paraffin section chemistry traditional technology, establishes one
Item being capable of the fast and convenient efficiently new technology for protein qualitative expression detection in the complex.
As shown in Figure 1, the preparation side of the novel C OCs composite construction tissue comprising multiple complexs of the embodiment of the present invention
Method the following steps are included:
S101: using suction or post-mortem method, cumulus oocytes complesxes materials: are obtained from folliculus ovarii
(COCs)；
S102: fixed: COCs being placed in 4% paraformaldehyde solution and fixes 2 hours；
S103: dehydration: COCs is respectively incubated for 20 minutes in 50% alcohol and dehydrated alcohol；
S104: transparent: by COCs in solution (dimethylbenzene+dehydrated alcohol 1:1) transition, to be transferred in 100% dimethylbenzene liquid and incubate
It educates 10 minutes；
S105: waxdip embedding: COCs is drawn into 1.5mlEP pipe, in 60 DEG C of dewaxing casees, is immersed with soft wax and hard wax
Each 30 minutes；The embedding case of injection 1CM square is sucked out, is embedded；
S106: slice and patch: embedded wax stone is fixed on slicer, is cut into a thickness of 5 μm of thin slices.It is attached to load
On slide, puts in 45 DEG C of insulating boxs and dry；
S107: it dewaxing: is dewaxed 10 minutes with dimethylbenzene；100% ethyl alcohol is incubated for 10 minutes；
S108: closing and antibody incubation: 5% secondary antibody source is added dropwise and closes serum, room temperature is closed 30 minutes in wet box；Primary antibody
4 DEG C of refrigerator overnights are incubated in wet box；Secondary antibody is after magazine is incubated at room temperature 1h, rinsing；
S109: it mounting observation: is observed after mounting with fluorescence microscope or confocal fluorescent microscopic.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of preparation method of the novel C OCs composite construction tissue comprising multiple complexs, which is characterized in that described to include
The preparation method of the novel C OCs composite construction tissue of multiple complexs is obtained from folliculus ovarii using suction or post-mortem method
Cumulus oocytes complesxes；Using cumulus oocytes complesxes as basic material, by the way of paraffin embedding, by ovum
Mound-oocyte complex is drawn into EP pipe, after fixation, washing, dehydration, transparent and waxdip step, is embedded；
The preparation method further includes slicing step, specifically:
Embedded wax stone is fixed on slicer, is thinly sliced, is attached on glass slide, puts in insulating box and dries；
It is dewaxed with dimethylbenzene, 100% ethyl alcohol is incubated for；
5% secondary antibody source closing serum is added dropwise, room temperature is closed 30 minutes in wet box；4 DEG C of refrigerator overnights are incubated in primary antibody wet box；Two
Resist after magazine is incubated at room temperature 1h, rinsing.
2. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as described in claim 1, feature
Be, wherein fixation, washing, dehydration, transparent, waxdip, embedding specific steps are as follows:
Step 1 obtains cumulus oocytes complesxes using suction or post-mortem method from folliculus ovarii；
Cumulus oocytes complesxes are placed in paraformaldehyde solution fixed by step 2；
Cumulus oocytes complesxes are dehydrated by step 3 in 50% alcohol and dehydrated alcohol；
Step 4 is transferred to transparent in dimethylbenzene liquid by cumulus oocytes complesxes after solution transition；Solution be dimethylbenzene+
Dehydrated alcohol 1:1；
Cumulus oocytes complesxes are drawn into 1.5mlEP pipe by step 5, in dewaxing case, are soaked with soft wax and hard wax
Enter；The embedding case of injection 1CM square is sucked out, is embedded.
3. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, this method uses fluorescence microscope after further comprising the steps of: mounting.
4. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, cumulus oocytes complesxes are placed in 4% paraformaldehyde solution by the step 2 fixes 2 hours.
5. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, the step 3, cumulus oocytes complesxes is respectively incubated for 20 minutes in 50% alcohol and dehydrated alcohol.
6. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, the step 4, by cumulus oocytes complesxes after solution transition, is transferred in 100% dimethylbenzene liquid transparent 10 points
Clock.
7. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, cumulus oocytes complesxes are drawn into 1.5mlEP pipe by the step 5, in 60 DEG C of dewaxing casees, use soft wax
It is immersed each 30 minutes with hard wax；The embedding case of injection 1CM square is sucked out, is embedded.
8. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, embedded wax stone is cut into a thickness of 5 μm of thin slices, is attached on glass slide, puts in 45 DEG C of insulating boxs and dries.
9. the preparation method of the novel C OCs composite construction tissue comprising multiple complexs as claimed in claim 2, feature
It is, is dewaxed 10 minutes with dimethylbenzene；100% ethyl alcohol is incubated for 10 minutes.
10. a kind of novel C OCs composite construction tissue comprising described in claim 1-9 any one including multiple complexs
The frozen section manufacture craft of preparation method preparation novel C OCs composite construction tissue.
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Citations (6)
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|---|---|---|---|---|
| CN101144760A (en) * | 2007-04-23 | 2008-03-19 | 中国农业大学 | Method for preparing oocyte paraffin cut film |
| CN101709290A (en) * | 2009-12-03 | 2010-05-19 | 安徽农业大学 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
| CN101851606A (en) * | 2010-04-07 | 2010-10-06 | 魏红江 | Cumulus oophorus-oocyte compound cutting filter method |
| CN102116711A (en) * | 2011-01-31 | 2011-07-06 | 山东东方海洋科技股份有限公司 | Manufacturing method of paraffin sections of zostera marina embryo |
| CN103091151A (en) * | 2013-01-04 | 2013-05-08 | 复旦大学附属中山医院 | Wax cell chip inoculation method |
| CN104931327A (en) * | 2015-03-24 | 2015-09-23 | 重庆医科大学 | Micron-sized biological material paraffin sectioning method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7732202B2 (en) * | 2005-10-21 | 2010-06-08 | International Stem Cell Corporation | Oxygen tension for the parthenogenic activation of human oocytes for the production of human embryonic stem cells |
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- 2016-07-01 CN CN201610515409.6A patent/CN106226133B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144760A (en) * | 2007-04-23 | 2008-03-19 | 中国农业大学 | Method for preparing oocyte paraffin cut film |
| CN101709290A (en) * | 2009-12-03 | 2010-05-19 | 安徽农业大学 | Simple, economic and efficient method for in-vitro maturity of porcine oocytes |
| CN101851606A (en) * | 2010-04-07 | 2010-10-06 | 魏红江 | Cumulus oophorus-oocyte compound cutting filter method |
| CN102116711A (en) * | 2011-01-31 | 2011-07-06 | 山东东方海洋科技股份有限公司 | Manufacturing method of paraffin sections of zostera marina embryo |
| CN103091151A (en) * | 2013-01-04 | 2013-05-08 | 复旦大学附属中山医院 | Wax cell chip inoculation method |
| CN104931327A (en) * | 2015-03-24 | 2015-09-23 | 重庆医科大学 | Micron-sized biological material paraffin sectioning method |
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