CN109991055A - The plasticity embedding method of multicolor fluorescence dyestuff and fluorescent protein labeling sample - Google Patents
The plasticity embedding method of multicolor fluorescence dyestuff and fluorescent protein labeling sample Download PDFInfo
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- 239000003112 inhibitor Substances 0.000 abstract description 2
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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
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
-
- 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
- G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Chemical & Material Sciences (AREA)
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- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Biomedical Technology (AREA)
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Present invention discloses a kind of suitable for multicolor fluorescence dyestuff and the plasticity embedding method of fluorescent protein labeling sample, comprising: pre-processes the biological organization sample of fluorescent marker；It is sequentially placed into gradient penetration in the resin solution of various concentration TB, each concentration 1h-2h permeates 12-24h in the resin solution of activity TB later；With resin penetration liquid to the fluorescent marker sample described above after the sample of gradient processing is embedded, heats polymerization, embedded.The present invention can inhibit bias light using TB as background inhibitor, and can be compatible with the use of multicolor fluorescence dyestuff and fluorescin.Embedding method provided by the present invention is suitable for various kinds of resin and embeds, while being suitable for the sample of a variety of fluorescent dyes and fluorescent protein labeling.
Description
Technical field
The present invention relates to biomedical engineering technology fields, and in particular to a kind of multicolor fluorescence dye marker sample plasticity packet
Bury method.
Background technique
Currently, fluorescin and fluorescence dye can be obtained simultaneously by fluorescin and fluorochrome label technology
Expect mark information, the relationships such as structure and function and disease to further investigation biological tissue have important role.
Existing embedding method embeds biological using pure resin or the resin sample infiltration for having dissolved SBB and DTT
Tissue can embed the biological tissue of multiple color fluorescent protein labeling, such as green/red/blue fluorescent protein label group
Knit, it is disadvantageous in that: this embedding method is unsuitable for various fluorochrome label biological samples, to greatly limit
It is applied, and for example, by using SBB as background inhibitor, but SBB has autofluorescence in red and infrared band, limits it more
Use in color especially red fluorescent protein and a variety of fluorescent dye samples.
Summary of the invention
The purpose of the present invention is overcoming the shortcomings of the prior art, provide a kind of suitable for a variety of fluorescent dyes and fluorescence
The plasticity embedding method of protein labeling sample.
In order to solve the above technical problems, technical method of the invention includes:
Biological organization sample is pre-processed；
Pretreated biological organization sample is sequentially placed into 0.It is permeated in the gradient concentration alcohol of 5-95wt%, Mei Geti
Degree 0.5-3 hours, then containing 0 respectively.It is permeated 6-24 hours in the gradient resin of 7-1wt/%TB different quality concentration；
The sample after gradient concentration permeates is embedded with resin penetration liquid, heats the fluorescence embedded after polymerization
Dye marker sample.
Preferably, it is described to biological sample carry out pretreatment include:
Biological sample is fixed 12-24 hours in paraformaldehyde fixer；
Biological sample after fixation is rinsed 6-12 hours in rinsing liquid；
Biological sample after the rinsing is sequentially placed into 50%, 70% ethanol solution of ice water precooling and carries out serial dehydration,
It is dehydrated in 95% or 100% ethanol solution again, 5 minutes to 2 hours every time.
Preferably, the mass concentration of the gradient resin of the different quality concentration is first gradient 40% ~ 60%, the second gradient
60% ~ 70%, 3rd gradient 70 ~ 90% and 4th gradient 90% ~ 100%.
Preferably, the mass concentration of the resin solution of the various concentration be respectively first gradient 50%, the second gradient 70%,
3rd gradient 85% and 4th gradient 100%.
Preferably, the mass concentration of the gradient concentration alcoholic solution is respectively 50%, 70%, 85% and 100%.
Preferably, described that the biological sample after gradient penetration is embedded with resin penetration liquid specifically: will
Biological sample after gradient penetration is placed in imbedded mold, is then full of embeding resin liquid in a mold.
Preferably, the resin in the resin penetration liquid and embeding resin solution is specially GMA resin.
Preferably, the fluorochrome label sample embedded after heating specifically: by the filled mold 35
It is heated 12 to 24 hours at DEG C -40 DEG C.
Preferably, the solvent of the resin solution is 95% ethyl alcohol.
Preferably, the biological sample is selected from the biological sample of fluorescence probe label.
The beneficial effects are mainly reflected as follows: the method for the present invention carries out infiltration using the resin containing TB and gathers with lower
It closes temperature and carries out embedding polymerization, can completely protect original fluorescence signal；It simultaneously can using the resin gradient penetration containing TB
The significant bias light for reducing red channel；The antigen active of biological tissue can be saved；Biological sample suitable for sizes
This；The biological tissue of obtained embedding can quickly obtain full brain in conjunction with Serial ultrathin sections and block face imaging technique simultaneously
The high-resolution distributed in three dimensions of fluorochrome label sample and fluorescent protein labeling sample, to carry out the solution of dependency structure and function
Analysis.
Detailed description of the invention
Technical scheme of the present invention is further explained with reference to the accompanying drawing:
Fig. 1 is that knot is imaged in brain tissue red fluorescence dyestuff (Dylight594) label vascular prepared by traditional resin embedding method
Fruit；
Fig. 2 is the red glimmering dyestuff of brain tissue of the resin embedding method preparation for the high s/n ratio that the embodiment of the present invention 1 embeds
(Dylight594) label vascular imaging results；
Fig. 3 is the brain tissue red fluorescence dyestuff of the resin embedding method preparation for the high s/n ratio that the embodiment of the present invention 1 embeds
(Dylight488) label vascular imaging results；
Fig. 4 is the brain tissue orchil (DANIR- of the resin embedding method preparation for the high s/n ratio that the embodiment of the present invention 2 embeds
8C) mark amyloid- β imaging results；
Fig. 5 is the brain tissue red fluorescent protein of the resin embedding method preparation for the high s/n ratio that the embodiment of the present invention 3 embeds
(tdTomato) imaging results；
Fig. 6 is the brain tissue green fluorescent protein of the resin embedding method preparation for the high s/n ratio that the embodiment of the present invention 3 embeds
(GFP) imaging results；
Fig. 7 is the brain tissue blue fluorescent protein of the resin embedding method preparation for the high s/n ratio that the embodiment of the present invention 3 embeds
(BFP) imaging results.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, With reference to embodiment
The present invention is described in further detail.
Explanation of nouns
GMA resin: glycidyl methacrylate.
TB: the trueblack resin of biotium company, U.S. production.
The present invention provides a kind of multicolor fluorescence dyestuff and the plasticity embedding methods of fluorescent protein labeling sample, comprising:
Biological organization sample is pre-processed；Pretreated biological organization sample is sequentially placed into 0.The gradient of 5-95wt%
It is permeated in concentration alcohol, each gradient 0.5-3 hours, then containing 0 respectively.The ladder of 7-1wt/%TB different quality concentration
It is permeated 6-24 hours in degree resin；The sample after gradient concentration permeates is embedded with resin penetration liquid, heat it is poly-
The fluorochrome label sample embedded after conjunction.The mass concentration of the gradient resin of the preferred different quality concentration is the
One gradient 40% ~ 60%, the second gradient 60% ~ 70%, 3rd gradient 70 ~ 90% and 4th gradient 90% ~ 100%.Described in preferred not
Mass concentration with the resin solution of concentration is respectively first gradient 50%, the second gradient 70%, 3rd gradient 85% and 4th gradient
100%.The mass concentration of the gradient concentration alcoholic solution is respectively 50%, 70%, 85% and 100%.
According to the present invention, the fluorochrome label sample that is embedded after heating specifically: by the filled mold
It is heated 12 to 24 hours at 35 DEG C -40 DEG C.
According to the present invention, the solvent of the resin solution is 95% ethyl alcohol.
For the biological sample preferably embedded, biological sample is pre-processed first, specifically: by biological sample
This fixes 12-24 hours in paraformaldehyde fixer；Biological sample after fixation is rinsed 6-12 hours in rinsing liquid；It will
Biological sample after the rinsing is sequentially placed into 50%, 70% ethanol solution of ice water precooling and carries out serial dehydration, then 95%
Or 100% be dehydrated in ethanol solution, 5 minutes to 2 hours every time.
It is described that the biological sample after gradient penetration is embedded with resin penetration liquid specifically:
Biological sample after gradient penetration is placed in imbedded mold, is then full of embeding resin liquid in a mold.The tree
Resin in rouge penetrating fluid and embeding resin solution is specially GMA resin.
All biological samples used are selected from the biological sample of fluorescence probe label in the present invention.
Technology incidence of criminal offenses of the invention is further described below by specific embodiment.
Embodiment 1
To the C57 mouse of adult, using tail vein injection DyLight594, is anaesthetized, adopted using 1% yellow Jackets after twenty minutes
It is irrigated and is fixed with heart reperfusion mode.First using the 0 of preheating.5-7min is perfused in 01M phosphate buffer,
Later using 4% paraformaldehyde and 2 of pre-cooling.5% sucrose mixed stationary liquid is perfused 7-10 minutes.It, quickly will be small after perfusion
Murine brain is removed and placed in the paraformaldehyde and 2 of 4% pre-cooling.In 5% sucrose mixed stationary liquid, rear fixed 6-24 in 4 DEG C of environment
Hour.
The brain tissue fixed afterwards uses the 0 of 4 DEG C of pre-coolings.01M phosphate buffer carries out rinsing 12-24 hours,
Fresh solution 3-5 times is replaced in centre, to completely remove fixer remaining in brain tissue.
After rinsing, brain tissue is put into 50%, 70% and 95% ethanol solution of pre-cooling and carries out serial dehydration, often
A gradient 1-2h.
After dehydration, brain tissue is put into pre-cooling and contains 0.In 50%, 70%, the 85% and 100% GMA resin of 5-1%TB
Carry out gradient penetration, each gradient 2-3 hour, wherein 50%, 70%, the solvent of 85%GMA resin penetration liquid be 95% ethyl alcohol；Then
Brain tissue after gradient penetration is put into containing 0.48-72h is permeated in the 100%GMA resin of 5%-1%TB, centre replacement is fresh
100%GMA resin 2 times.
Wherein, 100%GMA resin described above is by 52.3%GMA monomer, 3% water, 44.1% butyl methacrylate and 0.
6% azobisisoheptonitrile is formulated.
The brain tissue that infiltration terminates is put into polymerization mold, is filled it up in a mold containing 0.The GMA of 5%-1%TB is embedded
Liquid is placed in vacuum drying oven, vacuumizes the oxygen removed in mold, is adjusted baking oven to 38 DEG C, is polymerize 12-24 hours.Polymerization
After good, obtained brain tissue embedding sample is the fluorescent sample of high s/n ratio.
594 fluorescence imaging of Alex is carried out to embedded brain tissue, gained fluorescent image is referring to Fig. 2.
It using same embodiment, uses the brain tissue of DyLight488 label instead, the image of effect same can be obtained, join
According to Fig. 3.
Embodiment 2
The mouse brain for taking DANIR-8C to dye, is placed in the paraformaldehyde and 2 of 4% pre-cooling.In 5% sucrose mixed stationary liquid, 4 DEG C of environment
In after fixed 6-24 hours.
The brain tissue fixed afterwards uses the 0 of 4 DEG C of pre-coolings.01M phosphate buffer carries out rinsing 12-24 hours,
Fresh solution 3-5 times is replaced in centre, to completely remove fixer remaining in brain tissue.
After rinsing, brain tissue is put into 50%, 70% and 95% ethanol solution of pre-cooling and carries out serial dehydration, often
A gradient 1-2h.
After dehydration, brain tissue is put into pre-cooling and contains 0.In 50%, 70%, the 85% and 100% GMA resin of 5-1%TB
Carry out gradient penetration, each gradient 2-3 hour, wherein 50%, 70%, the solvent of 85%GMA resin penetration liquid be 95% ethyl alcohol；Then
Brain tissue after gradient penetration is put into containing 0.48-72h is permeated in the 100%GMA resin of 5%-1%TB, centre replacement is fresh
100%GMA resin 2 times.
Wherein, 100%GMA resin described above is by 52.3%GMA monomer, 3% water, 44.1% butyl methacrylate and 0.
6% azobisisoheptonitrile is formulated.
The brain tissue that infiltration terminates is put into polymerization mold, is filled it up in a mold containing 0.The GMA of 5%-1%TB is embedded
Liquid is placed in vacuum drying oven, vacuumizes the oxygen removed in mold, is adjusted baking oven to 38 DEG C, is polymerize 12-24 hours.Polymerization
After good, obtained brain tissue embedding sample is the fluorescent sample of high s/n ratio.
Fluorescence imaging is carried out to embedded brain tissue, gained fluorescent image is referring to Fig. 4.
Embodiment 3
It to the VIP::Ai14 mouse of adult, is anaesthetized, is irrigated using heart perfusion mode and solid using 1% yellow Jackets
It is fixed.First using the 0 of preheating.5-7min is perfused in 01M phosphate buffer, later using 4% paraformaldehyde and 2 of pre-cooling.
5% sucrose mixed stationary liquid is perfused 7-10 minutes.After perfusion, Mice brain tissues are quickly removed and placed in the more of 4% pre-cooling
Polyformaldehyde and 2.It is rear 6-24 hours fixed in 4 DEG C of environment in 5% sucrose mixed stationary liquid.
The brain tissue fixed afterwards uses the 0 of 4 DEG C of pre-coolings.01M phosphate buffer carries out rinsing 12-24 hours,
Fresh solution 3-5 times is replaced in centre, to completely remove fixer remaining in brain tissue.
After rinsing, brain tissue is put into 50%, 70% and 95% ethanol solution of pre-cooling and carries out serial dehydration, often
A gradient 1-2h.
After dehydration, brain tissue is put into pre-cooling and contains 0.In 50%, 70%, the 85% and 100% GMA resin of 5-1%TB
Carry out gradient penetration, each gradient 2-3 hour, wherein 50%, 70%, the solvent of 85%GMA resin penetration liquid be 95% ethyl alcohol；Then
Brain tissue after gradient penetration is put into containing 0.48-72h is permeated in the 100%GMA resin of 5%-1%TB, centre replacement is fresh
100%GMA resin 2 times.
Wherein, 100%GMA resin described above is by 52.3%GMA monomer, 3% water, 44.1% butyl methacrylate and 0.
6% azobisisoheptonitrile is formulated.
The brain tissue that infiltration terminates is put into polymerization mold, is filled it up in a mold containing 0.The GMA of 5%-1%TB is embedded
Liquid is placed in vacuum drying oven, vacuumizes the oxygen removed in mold, is adjusted baking oven to 38 DEG C, is polymerize 12-24 hours.Polymerization
After good, obtained brain tissue embedding sample is the fluorescent sample of high s/n ratio.
TdTomato fluorescence imaging is carried out to embedded brain tissue, gained fluorescent image is referring to Fig. 5.
Using same embodiment, uses the brain tissue of GFP/BFP label instead, the image of effect same, reference can be obtained
Fig. 6-7.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (10)
1. the plasticity embedding method of a kind of multicolor fluorescence dyestuff and fluorescent protein labeling sample characterized by comprising
Biological organization sample is pre-processed；
Pretreated biological organization sample is sequentially placed into 0；
It is permeated in the gradient concentration alcohol of 5-95wt%, each gradient 0；
5-3 hours, then containing 0 respectively；
It is permeated 6-24 hours in the gradient resin of 7-1wt/%TB different quality concentration；
The sample after gradient concentration permeates is embedded with resin penetration liquid, heats the fluorescence embedded after polymerization
Dye marker sample.
2. the method according to claim 1, wherein it is described to biological organization sample carry out pretreatment include:
Biological organization sample is fixed 12-24 hours in paraformaldehyde fixer；
Biological organization sample after fixation is rinsed 6-12 hours in rinsing liquid；
Biological organization sample after the rinsing is sequentially placed into progress gradient in 50%, 70% ethanol solution of ice water precooling to take off
Water, then be dehydrated in 95% or 100% ethanol solution, 5 minutes to 2 hours every time.
3. the method according to claim 1, wherein the mass concentration of the gradient resin of the different quality concentration
For first gradient 40% ~ 60%, the second gradient 60% ~ 70%, 3rd gradient 70 ~ 90% and 4th gradient 90% ~ 100%.
4. according to the method described in claim 3, it is characterized in that, the mass concentration of the resin solution of the various concentration is distinguished
For first gradient 50%, the second gradient 70%, 3rd gradient 85% and 4th gradient 100%.
5. the method according to claim 1, wherein the mass concentration of the gradient concentration alcoholic solution is respectively
50%, 70%, 85% and 100%.
6. the method according to claim 1, wherein it is described with resin penetration liquid to described after gradient penetration
Biological organization sample is embedded specifically:
Biological organization sample after gradient penetration is placed in imbedded mold, is then full of embeding resin liquid in a mold.
7. according to the method described in claim 5, it is characterized in that, resin in the resin penetration liquid and embeding resin solution
Specially GMA resin.
8. the method according to claim 1, wherein the fluorochrome label sample embedded after heating is specific
Are as follows:
The filled mold is heated 12 to 24 hours at 35 DEG C -40 DEG C.
9. the method according to claim 1, wherein the solvent of the resin solution is 95% ethyl alcohol.
10. method described according to claim 1 ~ 9 any one claims, which is characterized in that the biological organization sample
Biological organization sample selected from fluorescence probe label.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111474358A (en) * | 2020-04-16 | 2020-07-31 | 武汉巴菲尔生物技术服务有限公司 | 3D (three-dimensional) immunofluorescence staining kit and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180321229A1 (en) * | 2017-05-04 | 2018-11-08 | Vector Laboratories, Inc. | Immunofluorescence assays |
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