CN106198990A - A kind of tissue samples is carried out immune labeled method - Google Patents
A kind of tissue samples is carried out immune labeled method Download PDFInfo
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- CN106198990A CN106198990A CN201510219570.4A CN201510219570A CN106198990A CN 106198990 A CN106198990 A CN 106198990A CN 201510219570 A CN201510219570 A CN 201510219570A CN 106198990 A CN106198990 A CN 106198990A
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Abstract
The invention discloses and a kind of tissue samples is carried out immune labeled method, specifically, the present invention (a) provides a labelling system, described labelling system to contain pending immune labeled tissue samples, for the probe being marked described tissue samples and buffer；(b) described labelling system is placed under electric field action it is marked process, so that described probe enters inside described tissue samples, thus described tissue samples is carried out immune labeled, it is thus achieved that through immune labeled tissue samples.The present invention can not only shorten probe and enter the time within tissue samples, moreover it is possible to keeps the integrity of organization internal, has great using value.
Description
Technical field
The invention belongs to biological technical field, in particular it relates to a kind of, tissue samples is carried out immune labeled side
Method.
Background technology
Research biomedical tissue, at cell and the three-D space structure of subcellular fraction yardstick, is to understand its normal function
Basis, also can provide foundation for grasping the generation of organ disease and evolution.In the past to people and other animal groups
The research of the research knitted predominantly anatomy yardstick, the research at cell and subcellular fraction yardstick is then limited to resolve energy
The restriction of power, is typically only capable to study the structural information of tissue slice.Three Dimensional Reconfiguration based on continuous tissue section
Research tissue is the most time-consuming and laborious.
The fast development of transparency of organization technology in recent years makes people obtain the high-resolution three-dimension of complete bio tissue
Structure is possibly realized, and is most commonly used that CLARITY technology at present, and it is by the crosslinking by hydrogel (Hydrogel)
The polymer formed is fixed with the biomolecule (albumen and DNA etc.) in tissue, and by using lauryl sulphate acid
Sodium (SDS) removes cell membrane etc. and has the biomolecule of very strong scattering to light, can ensure that organizational structure is not broken
On the premise of Huai, quickly by biological tissue's transparence, and realize the degree of depth (～6mm) three-dimensional imaging of complete tissue.
CLARITY technology is applied to transparence and the structural research of Mice brain tissues the earliest, and is progressively extended to whole little
Major organs (kidney, liver etc.) in Mus, illustrates this technology and is obtaining biological tissue's Complete three-dimensional high-resolution knot
The huge potential value of structure message context.
But, when being used for studying complete tissue three-dimensional high resolution structures by CLARITY technology, label probe is (special
It is not antibody) need long time from the surface arrival organization internal of complete tissue, hinder this technology greatly
Promotion and application.Such as, for the intact mice brain samples that 5mm is thick, an intact immune labelling needs
The time of at least 1.5 months just can complete.
Therefore, this area in the urgent need to exploitation a kind of can high degree shorten tissue samples immunofluorescence mark
Between clocking, and the tachysynthesis labeling method of organization internal integrity can also be kept.
Summary of the invention
It is an object of the invention to provide a kind of can high degree shorten tissue samples immunofluorescence label time
Between, and the rocket immunofluorescent labeling method of organization internal integrity can also be kept.
A first aspect of the present invention provides and a kind of tissue samples carries out immune labeled method, including step:
A () provides a labelling system, described labelling system to contain pending immune labeled tissue samples, for right
Probe that described tissue samples is marked and buffer；With
B described labelling system is placed under electric field action and is marked process by (), so that described probe enters
Enter inside described tissue samples, thus described tissue samples is carried out immune labeled, it is thus achieved that through immune labeled tissue
Sample.
In another preference, in step (b), when the time that described labelling processes is 1min-5h, it is preferred that
20min-1h, more preferably, 30min.
In another preference, in step (b), the temperature that described labelling processes is 4-50 DEG C, it is preferred that 10-40
DEG C, more preferably, 37 DEG C.
In another preference, described method also includes: (c) is carried out through immune labeled tissue samples described
Detection.
In another preference, in step (c), described detection includes fluoroscopic examination.
In another preference, described electric field has the feature that voltage is 25V, and interelectrode distance is 2.2cm,
Electric field intensity is 11.3V/cm.
In another preference, the pH of described labelling system is 5-11.
In another preference, described probe includes: antibody, nucleic probe.
In another preference, described probe is with detecting mark.
In another preference, described detected mark includes: fluorogen, chromophore, chemiluminescence group.
In another preference, when described probe is antibody, the pH of described labelling system is 5-11.
In another preference, described antibody is monoclonal antibody.
In another preference, the isoelectric level (PI) of described monoclonal antibody is 6.4-9.0.
In another preference, described tissue samples is the tissue samples expressing endogenous fluorescence albumen.
In another preference, described fluorescin is GFP albumen.
In another preference, described tissue samples is cerebral tissue, gastric tissue, hepatic tissue, lung tissue or its group
Close.
In another preference, described tissue samples derives from mammal, people or a combination thereof.
In another preference, described tissue samples derives from mice, rat, people or a combination thereof.
In another preference, described tissue samples is the sample through Vitrification management.
In another preference, described sample is lamellar sample, has the first first type surface and the second first type surface.
In another preference, the thickness of described lamellar sample is 2-20mm, it is preferred that 3-18mm, more preferably
Ground, 5-10mm.
In another preference, the sectional area of described lamellar sample is 1-100cm2。
In another preference, described electric field is by being positioned at the described sample left and right sides or the electrode of upper and lower both sides
Apply.
In another preference, described electric field is by being positioned at the first first type surface and second first type surface of described sample
The electrode in outside applies.
In another preference, the described probe entrance time within tissue samples is 20min-1h, it is preferred that
30-50min, more preferably, 30-40min.
In another preference, the time that described probe enters within tissue samples shortens 800 than traditional method
Times.
In another preference, described is immune labeled for immunofluorescence label.
In another preference, described method is the in vitro method of nondiagnostic and non-therapeutic.
Second aspect present invention provides and a kind of tissue samples carries out immune labeled device, and described device includes:
For placing the container of described tissue samples；Wherein, described container is used for holding a labelling system, described
Labelling system contains pending immune labeled tissue samples, for the probe that described tissue samples is marked and
Buffer；
For producing the electrode pair of electric field, wherein, described electrode be pointed to described tissue samples the left and right sides or
Both sides up and down, thus produce the electric field within tissue samples driven described in the entrance of described probe；With
Power supply, described power supply with described electrode to electrically connecting.
In another preference, described container is circular.
In another preference, a diameter of 1-10cm of described container, it is preferred that 3-4cm, more preferably, 3.5cm.
In another preference, described electrode diameter is 0.1-1mm, it is preferred that 0.2-0.8mm, more preferably,
0.3-0.6mm；The a length of 2-15cm of described electrode, it is preferred that 4-10cm, more preferably, 5-9cm.
Third aspect present invention provides and a kind of tissue samples carries out immune labeled test kit, described test kit
Contain:
First container, containing pending immune labeled tissue samples in described container；
Second container, containing the probe being marked described tissue samples in described container；
3rd container, containing buffer in described container, the pH of described buffer is 5-11；
4th container, containing battery lead plate, attaching plug in described container, forming an electric field intensity (V/cm) is
The electric field of 5-15.
Label or description, described label or description indicate described test kit for exempting from tissue samples
Epidemic disease labelling.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (as implemented
Example) in can be combined with each other between each technical characteristic of specifically describing, thus constitute new or preferred skill
Art scheme.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 shows IgG motion conditions under electric field action.
Fig. 2 shows the fluorescent quantitation result of IgG in tissue gel composite.
Fig. 3 shows the YFP RST of Thy1-YFP mouse brain slices after the immunostaining of electric field.
Fig. 4 shows the anti-YFP immunostaining results of the Thy1-YFP mouse brain slices of electric field.
Detailed description of the invention
The present inventor is through extensively in-depth study, first it was unexpectedly observed that strong by regulation extra electric field
Degree, probe can be rapidly introduced into inside tissue samples, carries out immune labeled to tissue samples, and can protect
Holding the integrity of organization internal, specifically, when electric field strength (V/cm) is 5-15, probe is at 20min-1
Organization internal can be arrived within h, tissue is carried out immune labeled.The present invention can not only shorten probe and enter
Time within tissue samples, moreover it is possible to keep the integrity of organization internal, there is great using value.
Tissue samples is carried out immune labeled
As used herein, described " carrying out immune labeled to tissue samples " is often referred to go with antibody specific binding
A kind of labeling method of antigen, its utilize the specific binding characteristics of specific antibodies to separate antigen, guide antigen,
And/or quantitative antigen.
In the present invention, described " carrying out immune labeled to tissue samples " refers under the effect of extra electric field,
Carrying out immune labeled with probe to tissue samples inside, compared with traditional method, the method can not only be accelerated to visit
Pin enters inside tissue samples, moreover it is possible to keep the integrity within tissue samples.Specifically, at the work of extra electric field
Under with, probe can enter inside tissue samples in 20min-1h, shortens complete transparence group greatly
The time of active immunity labelling, compared with the method for conventional antibodies molecule diffusion, the time can be shortened 800 by the method
Times.
What the present invention provided carries out immune labeled method to tissue samples, comprises the steps:
A () provides a labelling system, described labelling system to contain pending immune labeled tissue samples, for right
Probe that described tissue samples is marked and buffer；
B described labelling system is placed under electric field action and is marked process by (), so that described probe enters
Enter inside described tissue samples, thus described tissue samples is carried out immune labeled, it is thus achieved that through immune labeled tissue
Sample.
Tissue samples is carried out immune labeled device
As used herein, described " tissue samples is carried out immune labeled device " including:
For placing the container of described tissue samples；Wherein, described container is used for holding a labelling system, described
Labelling system contains pending immune labeled tissue samples, for the probe that described tissue samples is marked and
Buffer；
For producing the electrode pair of electric field, wherein, described electrode be pointed to described tissue samples the left and right sides or
Both sides up and down, thus produce the electric field within tissue samples driven described in the entrance of described probe；With
Power supply, described power supply with described electrode to electrically connecting.
With " tissue samples is carried out immune labeled device " of the present invention, tissue samples is carried out immunity to mark
Note, can not only shorten probe and enter the time within tissue samples, moreover it is possible to keep the integrity within tissue samples.
Tissue samples is carried out immune labeled test kit
As used herein, described " tissue samples is carried out immune labeled test kit " including:
First container, containing pending immune labeled tissue samples in described container；
Second container, containing the probe being marked described tissue samples in described container；
3rd container, containing buffer in described container, the pH of described buffer is 5-11；
4th container, containing battery lead plate, attaching plug in described container, forming an electric field intensity (V/cm) is
The electric field of 5-15.
Label or description, described label or description indicate described test kit for exempting from tissue samples
Epidemic disease labelling.
" tissue samples is carried out immune labeled test kit " of the present invention can not only be quickly to tissue
Sample interior carries out immune labeled, the most easy to carry.
Main advantages of the present invention include:
(1) under the effect of extra electric field, probe can enter inside tissue samples in 20min-1h, pole
The big time shortening complete transparence histogenic immunity labelling.Compared with the method for conventional antibodies molecule diffusion, should
Time can be shortened 800 times by method.
(2) under the effect of extra electric field, the structure within tissue samples still keeps integrity.
(3) present invention expands the range of application of transparency of organization technology, and can realize large scale biology group
The research of the three-dimension high-resolution structural information knitted.
Below in conjunction with specific embodiment, state the present invention further.Should be understood that these embodiments are only used for
The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted detailed conditions in the following example
Method, generally according to normal condition such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring Harbor Laboratory Press, 1989) condition described in, or according to proposed by manufacturer
Condition.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Embodiment 1 prepares the cerebral tissue sample of transparence mice
The mouse brain slices razor blade going fat complete accomplishes rectangle, and is loaded between two panels coverslip.Adjust
The thickness of the blue fourth glue of joint, it is ensured that after loading, blue fourth glue can be by the lower edges closing of brain sheet for brain sheet.At two panels lid glass
Between sheet, the edge along blue fourth glue adds a small amount of epoxide-resin glue.
Embodiment 2 is the cerebral tissue sample configuration electric field arrangement of transparence mice
The slide made-brain sheet combination is put in culture dish, the lid of 35mm culture dish bores two directly
The duck eye of footpath 1mm, and by two a diameter of 0.5mm, long 8cm platinum electrode (being purchased from Sigma) from duck eye
In pass, electrode is bent into right angle, at duck eye, electrode is reinforced with blue fourth glue, add in culture dish
2ml antibody diluent, covers lid on culture dish, is loaded on microscope carrier by culture dish, uses crocodile
Clamp electrode and be connected with power supply (KXN-6020D, ZHAOXIN).
Embodiment 3 electric field acceleration antibody enters the testing result of cerebral tissue sample interior
Experimental technique:
1) IgG (being purchased from molecular phycobiliprotein complexes) of 6 μ l is diluted in the sodium borate buffer liquid of the 0.1M of 2000 μ l
(pH8.5) in.
2) antibody diluent is added in culture dish, cover electrode cap.
3) after finding the diffusion edge of sample under the microscope, Taking Pictures recording.
4) Taking Pictures recording antibody molecule spread condition in brain sheet again is stood after 30 minutes.
5) turning on the power, regulation voltage is to 25V, and electrophoresis is Taking Pictures recording after 30 minutes.
6) picture and the information of extraction are processed.
Experimental result:
(1) as shown in Figure 1-2, result shows, under the effect of electric field, antibody is more uniform is filled with whole brain
Sheet, compared with the matched group being not powered on field, antibody can be full of whole brain sheet in 30 minutes.
(2) if before antibody concentration is reached the diffusion that the tissue depth at the 50% of Cmax is defined as antibody
Edge, then IgG has about been diffused into the place that organization internal 3.9mm is deep in 30 minutes.Therefore, IgG molecule is certainly
It is about being not added with 800 times in the case of electric field by the time diffusing to identical diffusion front and needing.
Embodiment 4 carries out immunostaining to Mice brain tissues sample
Experimental technique:
1) the Thy1-YFP mouse brain slices going fat good is carried out mounting.
2) IgG antibody (anti-GFP antibody, be purchased from molecular phycobiliprotein complexes) by 6 μ l is diluted in the 0.1M of 2000 μ l
Sodium borate buffer liquid in (pH8.5), and be slowly added in culture dish.
3) turn on the power, regulation voltage to 25V, electrophoresis 30 minutes, help antibody to enter organization internal.
4) power supply is closed.Stationary incubation 90 minutes, allows antibody and antigen fully combine.
5) turn on the power.Unconjugated for organization internal antibody, to 25V, electrophoresis 30 minutes, is removed by regulation voltage.
6) suck the antibody diluent in culture dish, change 0.1M borate buffer clean for 2ml.
7) imaging, observes labelling result.
Experimental result:
As shown in Figure 3-4, experienced by before and after's electrophoresis of 60 minutes, the YFP signal in Thy1-YFP mouse brain slices depends on
Old protect by intact, and YFP signal can be good at and antibody signal overlaps.
Result shows, External electrical field can help IgG antibody to rapidly enter inside tissue samples, and to organization internal
Carry out immune labeled.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document
It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention,
The present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within this Shen equally
Please appended claims limited range.
Claims (10)
1. one kind carries out immune labeled method to tissue samples, it is characterised in that include step:
A () provides a labelling system, described labelling system to contain pending immune labeled tissue samples, for right
Probe that described tissue samples is marked and buffer；With
B described labelling system is placed under electric field action and is marked process by (), so that described probe enters
Enter inside described tissue samples, thus described tissue samples is carried out immune labeled, it is thus achieved that through immune labeled tissue
Sample.
2. the method for claim 1, it is characterised in that described method also includes: (c) is to described warp
Immune labeled tissue samples detects.
3. method as claimed in claim 2, it is characterised in that in step (c), described detection includes glimmering
Light detects.
4. the method for claim 1, it is characterised in that the pH of described labelling system is 5-11.
5. the method for claim 1, it is characterised in that described sample is lamellar sample, has first
First type surface and the second first type surface.
6. method as claimed in claim 5, it is characterised in that the sectional area of described lamellar sample is
1-100cm2。
7. the method for claim 1, it is characterised in that described electric field is left by being positioned at described sample
The electrode of right both sides or up and down both sides applies.
8. one kind carries out immune labeled device to tissue samples, it is characterised in that described device includes:
For placing the container of described tissue samples；Wherein, described container is used for holding a labelling system, described
Labelling system contains pending immune labeled tissue samples, for the probe that described tissue samples is marked and
Buffer；
For producing the electrode pair of electric field, wherein, described electrode be pointed to described tissue samples the left and right sides or
Both sides up and down, thus produce the electric field within tissue samples driven described in the entrance of described probe；With
Power supply, described power supply with described electrode to electrically connecting.
9. device as claimed in claim 8, it is characterised in that a diameter of 1-10cm of described container.
10. one kind carries out immune labeled test kit to tissue samples, it is characterised in that described test kit contains:
First container, containing pending immune labeled tissue samples in described container；
Second container, containing the probe for being marked described tissue samples in described container；
3rd container, containing buffer in described container, the pH of described buffer is 5-11；
4th container, containing battery lead plate, attaching plug in described container, forming an electric field intensity (V/cm) is
The electric field of 5-15.
Label or description, described label or description indicate described test kit for exempting from tissue samples
Epidemic disease labelling.
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| CN201510219570.4A CN106198990A (en) | 2015-04-30 | 2015-04-30 | A kind of tissue samples is carried out immune labeled method |
| CN202010407241.3A CN111579769B (en) | 2015-04-30 | 2015-04-30 | Method for performing immune labeling on tissue sample |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108627563A (en) * | 2017-03-17 | 2018-10-09 | 王志伟 | A kind of electrophoretic apparatus and electrophoresis system and transparence tissue mark method |
| CN113049804A (en) * | 2021-03-17 | 2021-06-29 | 上海交通大学 | Method for rapidly marking biological tissues |
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| CN1363006A (en) * | 1999-06-11 | 2002-08-07 | 分析科学公司 | Electropnoresis-assisted staining of materials |
| CN101370553A (en) * | 2006-02-11 | 2009-02-18 | 基因特伦尼克斯公司 | Device and method for single-needle in vivo electroporation |
| WO2015041755A1 (en) * | 2013-09-20 | 2015-03-26 | California Institute Of Technology | Methods for phenotyping of intact whole tissues |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2883030B1 (en) * | 2012-08-09 | 2022-08-31 | The Board of Trustees of the Leland Stanford Junior University | Methods and compositions for preparing biological specimens for microscopic analysis |
| CN104198234B (en) * | 2014-07-29 | 2017-02-15 | 中国科学院自动化研究所 | Method allowing complete organs to be transparent while reserving tissue texture structure and corresponding mixed solution |
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- 2015-04-30 CN CN202010407241.3A patent/CN111579769B/en active Active
- 2015-04-30 CN CN201510219570.4A patent/CN106198990A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1363006A (en) * | 1999-06-11 | 2002-08-07 | 分析科学公司 | Electropnoresis-assisted staining of materials |
| CN101370553A (en) * | 2006-02-11 | 2009-02-18 | 基因特伦尼克斯公司 | Device and method for single-needle in vivo electroporation |
| WO2015041755A1 (en) * | 2013-09-20 | 2015-03-26 | California Institute Of Technology | Methods for phenotyping of intact whole tissues |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108627563A (en) * | 2017-03-17 | 2018-10-09 | 王志伟 | A kind of electrophoretic apparatus and electrophoresis system and transparence tissue mark method |
| CN113049804A (en) * | 2021-03-17 | 2021-06-29 | 上海交通大学 | Method for rapidly marking biological tissues |
| CN113049804B (en) * | 2021-03-17 | 2022-09-09 | 上海交通大学 | Method for rapidly marking biological tissues |
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| CN111579769A (en) | 2020-08-25 |
| CN111579769B (en) | 2023-10-27 |
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Application publication date: 20161207 |