CN106866876A - A kind of embedding medium of smooth transparence biological tissue, embedding method and application - Google Patents
A kind of embedding medium of smooth transparence biological tissue, embedding method and application Download PDFInfo
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- CN106866876A CN106866876A CN201710209321.6A CN201710209321A CN106866876A CN 106866876 A CN106866876 A CN 106866876A CN 201710209321 A CN201710209321 A CN 201710209321A CN 106866876 A CN106866876 A CN 106866876A
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- biological tissue
- embedding
- embedding medium
- hours
- transparence
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Links
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- -1 two components of A Chemical compound 0.000 claims abstract description 13
- 125000004386 diacrylate group Chemical group 0.000 claims abstract description 10
- 125000003342 alkenyl group Chemical group 0.000 claims abstract description 8
- 239000003505 polymerization initiator Substances 0.000 claims abstract description 8
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 71
- 239000011347 resin Substances 0.000 claims description 43
- 229920005989 resin Polymers 0.000 claims description 43
- 230000000977 initiatory effect Effects 0.000 claims description 37
- 239000003999 initiator Substances 0.000 claims description 20
- 239000012466 permeate Substances 0.000 claims description 14
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- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical group N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 6
- 125000000400 lauroyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 6
- QIWKUEJZZCOPFV-UHFFFAOYSA-N phenyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OC1=CC=CC=C1 QIWKUEJZZCOPFV-UHFFFAOYSA-N 0.000 claims description 6
- INQDDHNZXOAFFD-UHFFFAOYSA-N 2-[2-(2-prop-2-enoyloxyethoxy)ethoxy]ethyl prop-2-enoate Chemical compound C=CC(=O)OCCOCCOCCOC(=O)C=C INQDDHNZXOAFFD-UHFFFAOYSA-N 0.000 claims description 5
- 229920000178 Acrylic resin Polymers 0.000 claims description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 5
- RZVINYQDSSQUKO-UHFFFAOYSA-N 2-phenoxyethyl prop-2-enoate Chemical class C=CC(=O)OCCOC1=CC=CC=C1 RZVINYQDSSQUKO-UHFFFAOYSA-N 0.000 claims description 4
- 230000003287 optical effect Effects 0.000 claims description 4
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 3
- YYPNJNDODFVZLE-UHFFFAOYSA-N 3-methylbut-2-enoic acid Chemical compound CC(C)=CC(O)=O YYPNJNDODFVZLE-UHFFFAOYSA-N 0.000 claims description 3
- 150000001252 acrylic acid derivatives Chemical class 0.000 claims description 3
- LSXWFXONGKSEMY-UHFFFAOYSA-N di-tert-butyl peroxide Chemical compound CC(C)(C)OOC(C)(C)C LSXWFXONGKSEMY-UHFFFAOYSA-N 0.000 claims description 3
- 150000001993 dienes Chemical class 0.000 claims description 3
- WRAQQYDMVSCOTE-UHFFFAOYSA-N phenyl prop-2-enoate Chemical compound C=CC(=O)OC1=CC=CC=C1 WRAQQYDMVSCOTE-UHFFFAOYSA-N 0.000 claims description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 2
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 2
- KILNKMKUNHCLRK-UHFFFAOYSA-N C(=O)(OC(C)C)OOC(=O)OC(C)C.C(C)(C)(C)OC(C(C)(C)C)=O Chemical compound C(=O)(OC(C)C)OOC(=O)OC(C)C.C(C)(C)(C)OC(C(C)(C)C)=O KILNKMKUNHCLRK-UHFFFAOYSA-N 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 2
- GCTPMLUUWLLESL-UHFFFAOYSA-N benzyl prop-2-enoate Chemical compound C=CC(=O)OCC1=CC=CC=C1 GCTPMLUUWLLESL-UHFFFAOYSA-N 0.000 claims description 2
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 2
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 claims description 2
- GJBRNHKUVLOCEB-UHFFFAOYSA-N tert-butyl benzenecarboperoxoate Chemical compound CC(C)(C)OOC(=O)C1=CC=CC=C1 GJBRNHKUVLOCEB-UHFFFAOYSA-N 0.000 claims description 2
- BEQKKZICTDFVMG-UHFFFAOYSA-N 1,2,3,4,6-pentaoxepane-5,7-dione Chemical compound O=C1OOOOC(=O)O1 BEQKKZICTDFVMG-UHFFFAOYSA-N 0.000 claims 1
- 150000001336 alkenes Chemical class 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 abstract description 15
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- 239000007850 fluorescent dye Substances 0.000 description 8
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 8
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- 230000018044 dehydration Effects 0.000 description 7
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- AOJOEFVRHOZDFN-UHFFFAOYSA-N benzyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC1=CC=CC=C1 AOJOEFVRHOZDFN-UHFFFAOYSA-N 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
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- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
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- 239000004342 Benzoyl peroxide Substances 0.000 description 1
- UWIULCYKVGIOPW-UHFFFAOYSA-N Glycolone Natural products CCOC1=C(CC=CC)C(=O)N(C)c2c(O)cccc12 UWIULCYKVGIOPW-UHFFFAOYSA-N 0.000 description 1
- 241000165940 Houjia Species 0.000 description 1
- YIVJZNGAASQVEM-UHFFFAOYSA-N Lauroyl peroxide Chemical compound CCCCCCCCCCCC(=O)OOC(=O)CCCCCCCCCCC YIVJZNGAASQVEM-UHFFFAOYSA-N 0.000 description 1
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- JMPVESVJOFYWTB-UHFFFAOYSA-N dipropan-2-yl carbonate Chemical class CC(C)OC(=O)OC(C)C JMPVESVJOFYWTB-UHFFFAOYSA-N 0.000 description 1
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- 150000002825 nitriles Chemical class 0.000 description 1
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- QMMOXUPEWRXHJS-UHFFFAOYSA-N pent-2-ene Chemical group CCC=CC QMMOXUPEWRXHJS-UHFFFAOYSA-N 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001228 polyisocyanate Polymers 0.000 description 1
- 239000005056 polyisocyanate Substances 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- VXHFNALHLRWIIU-UHFFFAOYSA-N tert-butyl 2,2-dimethylpropanoate Chemical compound CC(C)(C)OC(=O)C(C)(C)C VXHFNALHLRWIIU-UHFFFAOYSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/30—Esters containing oxygen in addition to the carboxy oxygen containing aromatic rings in the alcohol moiety
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/12—Esters of monohydric alcohols or phenols
- C08F220/16—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms
- C08F220/18—Esters of monohydric alcohols or phenols of phenols or of alcohols containing two or more carbon atoms with acrylic or methacrylic acids
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/30—Esters containing oxygen in addition to the carboxy oxygen containing aromatic rings in the alcohol moiety
- C08F220/301—Esters containing oxygen in addition to the carboxy oxygen containing aromatic rings in the alcohol moiety and one oxygen in the alcohol moiety
Abstract
The invention discloses a kind of embedding medium and its embedding method of smooth transparence biological tissue.Embedding medium is high index of refraction acrylate, including two components of A, B；Component A include by weight, 90 to 100 parts of acrylate resin monomer, 0 to 10 part of diacrylate crosslinking agents；B component include by weight, the alkenyl polymerization initiator less equal than 10 parts；The weight proportion of component A and B component is 90:10 to 999:Between 1.Biological tissue is embedded using embedding medium of the invention, the light transparence degree of biological tissue is high, method is simple to operation, the fluorescence of Intrinsic fluorescence albumen and extrinsic fluorescence dyestuff keeps effect good, embedding success rate is high, mating plate fluorescence microscope and wide field fluorescence microscope can be applied, electron microscope can be also potentially applied to.
Description
Technical field
The invention belongs to bio-imaging field, more particularly, to a kind of embedding medium of smooth transparence biological tissue, embedding
Method and application.
Background technology
The network structure of cranial nerve is studied for treating the tool such as the mental illness of the mankind and the cognitive and emotion of the research mankind
It is of great importance, the resolution ratio for obtaining the 3D neural network structures of full brain scope and reaching cynapse rank is one for fluorescence imaging
Individual cross-cutting huge challenge, while can also promote the technological progress of following whole sciemtifec and technical sphere, particularly from complete system
The relation between brain structure and function is studied in function of organization.Realize studying biological tissue in complete biosystem
Still there is very big difficulty in individual molecule structural information.When biological tissue for fluorescence probe mark carries out optical imagery,
The mainstream technology to large sample imaging external at present is, by biological tissue's transparence combination mating plate fluorescence microscope imaging technique, to lead to
Crossing will realize that bilateral light excites fluorescence molecule to realize successively being imaged after biological tissue's transparence, this method image taking speed is fast, but
Be the current liquid reagent transparence sample making technology for using species it is various, respectively have advantage and disadvantage.
Refraction coefficient (RIs) of the light between different materials is different, causes scattered power of the light in system and increases
Greatly.It is exactly the light scattering effect for making imageable target reach minimum that light is transparent, and lipid is cause mammal brain light scattering main
Factor.Therefore, the refraction coefficient for removing the lipid on cell membrane and matching lipid surrounding biological tissue can just realize biological group
Knit transparent.Existing biological tissue's transparence technology is divided into chemical method and physics and two kinds of means of chemical bond.Using chemistry
The method of reagent can be divided into aqueous transparent reagent and oiliness transparence reagent again.Aqueous transparent reagent preserves fluorescin
Effect preferably, the treatment to biological tissue is more gentle, but the transparence speed of aqueous transparent reagent is relatively low, and transparence
The size of biological tissue easily expands afterwards, has influence on the analysis to biological organizer beginning pattern.Oiliness transparence reagent it is transparent
Change speed very fast, transparence degree is high and change in size of biological tissue is smaller, but oiliness reagent is to endogenous fluorescin
Be quenched it is larger.Physics is to combine hydrogel crosslinked bio tissue with electric field means with chemically combined method, is used
Water-based reagent accelerates to go to biological tissue lipoprotein function and realize transparence in the presence of electric field, the operating process of this method
Complexity, repeatability is poor, and requirement to equipment and personnel is high.Meanwhile, there is certain toxicity liquid light transparent agent more, prepare sample
This program is various, and specimen storage is more difficult and its fluorescence signal is difficult to long-term holding.The transparent metaplasia of liquid reagent of prior art
The reagent and transparence method of thing tissue more or less have a certain degree of destruction to the Intrinsic fluorescence albumen of biological tissue,
Fluorescence can not well keep.
In addition, in order to meet cranial nerve network structure imaging, only relying on biological tissue's endogenous fluorescence albumen and tending not to completely
The requirement of sufficient fluorescence imaging, resin embedding is realized by using external source fluorescent dye immunohistochemical markers simultaneously, strengthens fluorescence, from
And increase imaging effect, but the embedding medium that the resin embedding technology of prior art is used tends not to ensure external source fluorescent dye
Molecular fluorescence keeps good, therefore, needing one kind badly can keep endogenous fluorescence effect good, being capable of compatible external source fluorescent dye mark
Note, realizes that specimen storage is simple and can for a long time keep biological tissue's transparence technology of fluorescence signal and process is simple.
The content of the invention
For the disadvantages described above or Improvement requirement of prior art, the invention provides a kind of bag of smooth transparence biological tissue
Agent and methods and applications are buried, its object is to pass through embedding medium of the selection with appropriate index, to pretreated biological group
Knit or the biological tissue of external source fluorochrome label embedded, realize the transparence of biological tissue, at the same endogenous fluorescence and
External source fluorescence keeps good, thus solves chemical reagent and the transparence side of existing liquid reagent transparence biological tissue
There is fluorescent protein labeling in method and fluorochrome label is difficult to compatible, specimen storage inconvenience and fluorescence signal is difficult to long-term holding
Technical problem.
To achieve the above object, according to one aspect of the present invention, there is provided a kind of embedding of smooth transparence biological tissue
Agent, the embedding medium biological tissue is embedded after formed solidified resin, the solidified resin with described biological group
Knit with the refractive index for matching, so that the biological tissue realizes light transparence.
Preferably, the refractive index of the monomer of the embedding medium is between 1.46~1.55.
Preferably, the refractive index of the monomer of the embedding medium is 1.48~1.53.
Preferably, the embedding medium is resin embedding agent.
Preferably, the resin embedding agent is acrylic resin.
Preferably, the acrylic resin includes two components of A, B, the component A by weight, including 90 to 100
The acrylate resin monomer and 0 to 10 part of diacrylate crosslinking agents of part；The B component by weight, including is less than
Or the alkenyl polymerization initiator equal to 10 parts；The weight proportion of the component A and B component is 90:10 to 999:Between 1.
Preferably, the acrylate resin monomer contains the allyl compound of phenyl for side base.
Preferably, the acrylate resin monomer is 2- phenoxyethyl acrylates, phenyl methacrylate, propylene
One or more in acid benzyl ester, benzyl methacrylate and/or phenyl acrylate.
Preferably, the acrylate resin monomer is 2- phenoxyethyl acrylates and/or phenyl methacrylate.
Preferably, the diacrylate acid esters crosslinking agent is the acrylate compounds containing diene base.
Preferably, the diacrylate acid esters crosslinking agent is ethylene glycol dimethacrylate, triethylene glycol diacrylate
Ester, diethylene glycol double methacrylate, TEG dimethylacrylate, tetraethylene glycol diacrylate and/or triethylene glycol two
One or more in methacrylate.
Preferably, the diacrylate acid esters crosslinking agent is tetraethylene glycol diacrylate and/or triethylene glycol dimethyl propylene
Olefin(e) acid ester.
Preferably, the alkenyl polymerization initiator is azo-initiator and/or peroxide initiator, and the azo draws
Hair agent is the one of azodiisobutyronitrile, tert-butyl azodicarboxylate, ABVN and/or azo-bis-iso-dimethyl
Plant or various.
Preferably, the alkenyl polymerization initiator is azodiisobutyronitrile and/or ABVN；The peroxide draws
Hair agent is dibenzoyl peroxide, peroxidating double lauroyl, di-t-butyl peroxide, cumyl peroxide perbenzoic acids
The tert-butyl ester, peroxidating trimethylacetic acid tertiary butyl ester di-isopropyl peroxydicarbonate and/or di-cyclohexylperoxy di-carbonate are a kind of
Or it is various.
Preferably, the peroxide initiator is dibenzoyl peroxide and/or the double lauroyl of peroxidating.
It is according to another aspect of the present invention, there is provided a kind of embedding method of smooth transparence biological tissue including following
Step：
(1) biological tissue's pretreatment：Biological tissue is fixed and dewater treatment, pretreated biological tissue is obtained
Sample；
(2) permeate：The pretreated biological tissue that will be obtained in step (1) soaks at infiltration temperature and light protected environment
In described embedding medium, the embedding medium is set fully to permeate biological tissue, the biological tissue after being permeated；The infiltration temperature
Degree is preferably -10 DEG C to 4 DEG C；
(3) solidify：Biological tissue after the infiltration that will be obtained in step (2) is solidified according to the heating schedule of setting.
Preferably, step (3) heating schedule for setting as：Trigger solidification at being 40 DEG C to 55 DEG C in initiation temperature,
Hardening time is 12 hours to 36 hours.
Preferably, the heating schedule of the setting carries out solidification and concretely comprises the following steps：Solidification 8 is small at 40 DEG C of initiation temperature
When, then solidify 12 hours at 45 DEG C of initiation temperature, then solidify 6 hours at 50 DEG C of initiation temperature, finally triggering temperature
Solidify 3 hours at 55 DEG C of degree.
According to another aspect of the present invention, there is provided a kind of application of described light transparence biological tissue embedding agent,
It is applied to the optical imagery of biological tissue.
In general, by the contemplated above technical scheme of the present invention compared with prior art, can obtain down and show
Beneficial effect.
(1) the biotissue optical clearing method that the present invention is provided uses high index of refraction acrylate embedding medium, by solid
Resin realizes the fast transparent of biological tissue with the index matching of biological tissue after change；
(2) transparentization of the invention is simple, reagent that is using is cheap and easily-available；
(3) method of the light transparence biological tissue that the present invention is provided, goes for mating plate fluorescence microscope and machinery
Chromatography combines wide field fluorescence microscope, can also potentially be applied to Electronic Speculum.
(4) method of the light transparence biological tissue that the present invention is provided is applied to Intrinsic fluorescence albumen and extrinsic fluorescence
The transparence of dye marker biological tissue, is kept for the fluorescence signal time long, it is possible to achieve the fast imaging of biological tissue.
Brief description of the drawings
Fig. 1 is the flow of resin embedding biological tissue transparence method of the present invention；
Fig. 2 is the photo that embodiment 1 embeds biological tissue；
Fig. 3 is that embodiment 2 embeds front and rear biological tissue's Intrinsic fluorescence Protein G FP fluorescence intensities contrast；
Fig. 4 is that embodiment 3 embeds the front and rear fluorescence intensities of biological tissue's extrinsic fluorescence dyestuff Alexa 488 contrast；
Fig. 5 is the 3D neural network structures of the endogenous GFP marks biological tissue of the embedding of embodiment 4；
Fig. 6 is that embodiment 5 embeds front and rear biological tissue's extrinsic fluorescence dyestuff CY3 fluorescence intensities contrast；
Fig. 7 is the photo that comparative example 6 embeds biological tissue.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as additionally, technical characteristic involved in invention described below each implementation method
Not constituting conflict each other can just be mutually combined.
The embedding medium of smooth transparence biological tissue proposed by the present invention, embedding medium is resin embedding agent, such as be acrylic acid
Resin, acrylic resin includes two components of A, B：
Component A by weight, including 90 to 100 parts of acrylate resin monomer and 0 to 10 part of diacrylate
Crosslinking agent；
B component by weight, including the alkenyl polymerization initiator less equal than 10 parts；
The weight proportion of component A and B component is 90:10 to 999:Between 1.
Embedding medium forms solidified resin, the refraction of the monomer of embedding medium after being embedded to pretreated biological tissue
Rate can between 1.46~1.55, preferably 1.48~1.53, the refractive index of biological tissue is about 1.55-1.56, so, when
After the embedding medium is embedded to biological tissue, the solidified resin formed after the polymerization of embedding medium monomer has phase with biological tissue
The refractive index of matching, so can be achieved with the light transparence of biological tissue, it is mentioned here match refer to refractive index it is identical or
Person's difference is no more than 0.1.
Wherein, acrylate resin monomer contains the allyl compound of phenyl for side base, such as be 2- Phenoxyethyls
One or more in acrylate, phenyl methacrylate, benzyl acrylate and/or phenyl acrylate, preferably 2- benzene oxygen
Base ethyl propylene acid esters and/or phenyl methacrylate.The acrylate monomer that this side base contains phenyl has folding higher
Rate is penetrated, the refractive index of resin monomer can be on the whole improved, the refractive index after resin monomer polymerization can be improved further.
Diacrylate acid esters crosslinking agent can be the acrylate compounds containing diene base, preferably ethylene glycol dimethyl
Acrylate, triethylene glycol diacrylate, diethylene glycol double methacrylate, TEG dimethylacrylate, tetraethylene glycol
One or more in diacrylate and/or TEGDMA, preferably tetraethylene glycol diacrylate and/or
TEGDMA.It is this to be formed solidified resin as crosslinking agent containing double propenyl ester compound
Cross-linked structure, improves the mechanical property of resin.
Alkenyl polymerization initiator is azo-initiator and/or peroxide initiator, and azo-initiator can be azo
One or more of bis-isobutyronitrile, tert-butyl azodicarboxylate, ABVN and/or azo-bis-iso-dimethyl,
Preferably azodiisobutyronitrile and/or ABVN；Peroxide initiator can be double for dibenzoyl peroxide, peroxidating
Lauroyl, di-t-butyl peroxide, cumyl peroxide peroxidized t-butyl perbenzoate, peroxidating trimethylacetic acid tertiary butyl ester mistake
Aoxidize one or more of two diisopropyl carbonates and/or di-cyclohexylperoxy di-carbonate, preferably dibenzoyl peroxide and/
Or the double lauroyl of peroxidating.Polymerization temperature and fluorescence intensity when the selection of initiator affects embedding, the bag that the present invention is provided
Burying agent can trigger in middle low temperature, it is ensured that fluorescin activity is so as to ensure fluorescence intensity.
The embedding of biological tissue is carried out using above-mentioned embedding medium and the light transparence method of biological tissue is realized, including it is following
Step：
(1) biological tissue's pretreatment：Biological tissue is fixed and dewater treatment, pretreated biological tissue is obtained
Sample；
(2) permeate：The pretreated biological tissue that will be obtained in step (1) is in certain infiltration temperature and light protected environment
Under be immersed in the embedding medium as described in claim 1~7 any one, the embedding medium is fully permeated biological tissue, obtain
Biological tissue after must permeating；
(3) solidify：Biological tissue after the infiltration that will be obtained in step (2) is solidified according to the heating schedule of setting.
Wherein, step (2) the infiltration temperature is -10 DEG C to 4 DEG C.
Step (3) heating schedule for setting as：In initiation temperature for 40 DEG C to 55 DEG C initiations solidify, hardening time is
12 hours to 36 hours；The heating schedule of setting carries out solidification and concretely comprises the following steps：Solidify 8 hours at 40 DEG C of initiation temperature, so
Solidify 12 hours at 45 DEG C of initiation temperature afterwards, then solidify 6 hours at 50 DEG C of initiation temperature, finally in 55 DEG C of initiation temperature
Lower solidification 3 hours.
The embedding medium and method of the light transparence biological tissue that the present invention is provided, mainly with the acrylic acid of high index of refraction
Its refractive index is further improved after polyisocyanate polyaddition, when the refractive index 1.56 of the biological tissue after with dehydration matches, biological tissue
Transparence will be realized.The refractive index of common acrylate monomer is 1.45 or so, aggregates into the refractive index of solid resin only
Have 1.50 or so, it is impossible to which transparence is carried out to biological tissue.Polymerized monomer, crosslinking agent by selecting appropriate index of the invention
With initiator, the composition proportion of component A and B component in allotment embedding medium, allocate monomer refractive index be allowed to after polymerization just with
The index matching of the biological tissue after dehydration, it is 1.50 or so liquid methacrylate monomer to use refractive index, is aggregated into solid
Refractive index after state resin is 1.56 or so.The refractive index of this monomer it is higher because side base contain phenyl ring improve light lead to
Cross the index of refraction of resin.The method of biological tissue's transparence of the invention is simple to operation, Intrinsic fluorescence albumen and exogenous
The fluorescence of fluorescent dye keeps effect good, and embedding success rate is high, it is adaptable to the mating plate microscope of biological tissue's piece and large sample into
Picture, while the large sample automation for applying also for automatic cutting system and fluorescence microscope GC-MS is successively imaged, it is also possible to
It is potential to be applied to Electronic Speculum imaging.
The reaction principle of high refractive index monomers embedding biological tissue is as follows：
The embedding medium that light transparence biological tissue of the present invention uses is the embedding medium of response type crosslinked bio tissue, using this
The embedding medium of response type crosslinked bio tissue, its autofluorescence background is low, light transmittance is high, embedding process is simple, resin shrinkage rate
It is low and can preferably keep neural fine structure, have the advantage of epoxy resin and acrylate concurrently, it is applicable biological tissue
Plasticity is embedded, and success rate is high, and the embedding for being particularly well-suited to substantially product sample and the full brain of mouse brain cuts into picture.
It is below embodiment：
Embodiment 1 and 2 and 4：
A kind of embedding method of the embedding medium of biological tissue, is middle low-temperature setting embedding method, and Fig. 1 is biological resin embedding
The flow of transparency of organization method, as shown in figure 1, comprising the following steps：
(1) biological tissue's pretreatment：First, will take out fixed after being placed in 4% paraformaldehyde (PFA) after mouse brain perfusion
24 hours；Then rinsed 3 times, every time 8 hours using PBS solution.Again using tetrahydrofuran/distilled water solution in 4 DEG C of environment
It is dehydrated according to gradient, tetrahydrofuran/2 hour+100% of+75% tetrahydrofuran/2 hour of 50% tetrahydrofuran/2 hour+95
Tetrahydrofuran/4 hour of tetrahydrofuran/2 hour+100%；
(2) permeate：Then the mouse brain after dehydration is immersed in 2 hours in the resin monomer got ready under 4 DEG C of light protected environments
Liquid is changed again, is then impregnated again 2 days；
(3) solidify：Embedding medium is as shown in table 1, by weight, by the acrylate monomer 2- phenoxy group second shown in table 1
The mixture of 10 parts of 90 parts of base acrylate and crosslinking agent TEGDMA and and the isobutyl of initiator azo two
10 parts of nitrile compares 99 according to weight:After 1 mixing, carried out under certain flow velocity advertising 30 minutes with nitrogen, the resin that will be mixed
Monomer storage is standby in -30 DEG C of refrigerator；
After the spare resin monomer that the mouse brain that will finally permeate is placed in capsule and addition is mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, imaging is carried out by it is slowly dropped to after room temperature cooling, wherein solid
Change condition and concretely comprise the following steps：Solidify 8 hours at 40 DEG C of initiation temperature, then solidify 12 hours at 45 DEG C of initiation temperature,
Then solidify 6 hours at 50 DEG C of initiation temperature, finally solidify 3 hours at 55 DEG C of initiation temperature.
Embodiment 2
A kind of embedding method of the embedding medium of biological tissue, comprises the following steps：
Step (1) and step (2) are with embodiment 1.
(3) solidify：Embedding medium is as shown in table 1, by weight, by the acrylate monomer methacrylic acid shown in table 1
The mixture of 5 parts of 95 parts of phenyl ester and crosslinking agent tetraethylene glycol diacrylate with and 1 part of initiator ABVN according to weight
Than 99:After 1 mixing, carried out under certain flow velocity advertising 30 minutes with nitrogen, the resin monomer that will be mixed is stored in -30 DEG C
Refrigerator in it is standby；
After the spare resin monomer that the mouse brain that will finally permeate is placed in capsule and addition is mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, imaging is carried out by it is slowly dropped to after room temperature cooling, wherein solid
Change condition and concretely comprise the following steps：Solidify 8 hours at 40 DEG C of initiation temperature, then solidify 12 hours at 45 DEG C of initiation temperature,
Then solidify 6 hours at 50 DEG C of initiation temperature, finally solidify 3 hours at 55 DEG C of initiation temperature.
Embodiment 3
(1) biological tissue's pretreatment：First, will take out fixed after being placed in 4% paraformaldehyde (PFA) after mouse brain perfusion
24 hours；Mouse brain is carried out into bobbing machine again carries out slicing treatment, is then rinsed 3 times, every time 8 hours using PBS solution.Will be fixed
Immunohistochemical markers are carried out using organic fluorescent dye molecule Alexa 488 with the biological tissue after rinsing.20% is used first
The PBS solution of DMSO and 0.2%Triton X-100 carries out punching treatment 12 hours, Ran Houjia to 100 microns of murine brain pieces
The serum for entering 10% is closed 12 hours, according still further to 500 after rinsing 1 hour/3 times using PBS:1 ratio adds primary antibody at 37 DEG C
Vibrated under lucifuge and be incubated 2 days, vibrated under 37 DEG C of lucifuges after being incubated 2 days according still further to 800:1 ratio is shaken under adding 4 DEG C of lucifuges of secondary antibody
Dynamic incubation is rinsed 1 hour/3 times after 8 hours using PBS.
(2) permeate：Brain piece after fluorescent dye SABC is with 100% tetrahydrofuran/10 minute；Then by after dehydration
Brain piece is placed in resin monomer and permeates 10 minutes, is placed on slide, after dripping upper 2 drop resin monomer, covered；Wherein set
Alicyclic monomer is：According to the proportioning in table 1, by weight, by 95 parts of phenyl methacrylate, triethylene glycol dimethacrylate
After the mixing of 5 parts of ester, and 5 parts of initiator dilauroyl peroxide, according to 995:After 5 prepare, with nitrogen under certain flow velocity
Advertise 30 minutes, the resin monomer that will be mixed is stored in standby in -30 DEG C of refrigerator；
(3) solidify：The brain piece that will be sealed is placed in baking oven in vacuum drying oven, and it is carried out according to the condition of cure of setting
Solidification, imaging is carried out by it is slowly dropped to after room temperature cooling, wherein condition of cure and is concretely comprised the following steps：Triggering temperature
Solidify at 40 DEG C of degree 8 hours, then solidify 12 hours at 45 DEG C of initiation temperature, then solidification 6 is small at 50 DEG C of initiation temperature
When, finally solidify 3 hours at 55 DEG C of initiation temperature.
Embodiment 4
A kind of embedding method of the embedding medium of biological tissue, comprises the following steps：
Step (1) and step (2) are with embodiment 1.
(3) solidify：Embedding medium is as shown in table 1, by weight, by the acrylate monomer 2- phenoxy group second shown in table 1
100 parts of base acrylate compares 99 with 10 parts of initiator benzoyl peroxide according to weight:After 1 mixing, with nitrogen in certain stream
Carry out advertising 30 minutes under speed, the resin monomer that will be mixed is stored in standby in -30 DEG C of refrigerator；
After the spare resin monomer that the mouse brain that will finally permeate is placed in capsule and addition is mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, imaging is carried out by it is slowly dropped to after room temperature cooling, wherein solid
Change condition and concretely comprise the following steps：Solidify 8 hours at 40 DEG C of initiation temperature, then solidify 12 hours at 45 DEG C of initiation temperature,
Then solidify 6 hours at 50 DEG C of initiation temperature, finally solidify 3 hours at 55 DEG C of initiation temperature.
Embodiment 5：
(1) biological tissue's pretreatment：First, will take out fixed after being placed in 4% paraformaldehyde (PFA) after mouse brain perfusion
24 hours；Mouse brain is carried out into bobbing machine again carries out slicing treatment, is then rinsed 3 times, every time 8 hours using PBS solution.Will be fixed
Immunohistochemical markers are carried out using organic fluorescent dye molecule CY3 with the biological tissue after rinsing.First using 20%DMSO and
The PBS solution of 0.2%Triton X-100 carries out punching treatment 12 hours to 100 microns of murine brain pieces, is subsequently adding 10%
Serum close 12 hours, using PBS rinse 1 hour/3 times after according still further to 500:1 ratio adds primary antibody under 37 DEG C of lucifuges
Vibration is incubated 2 days, is vibrated under 37 DEG C of lucifuges after being incubated 2 days according still further to 800:1 ratio vibrates incubation under adding 4 DEG C of lucifuges of secondary antibody
Rinsed 1 hour/3 times using PBS after 8 hours.
(2) permeate：Brain piece after fluorescent dye SABC is with 100% tetrahydrofuran/10 minute；Then by after dehydration
Brain piece is placed in resin monomer and permeates 10 minutes, is placed on slide, after dripping upper 2 drop resin monomer, covered；Wherein set
Alicyclic monomer is：According to the proportioning in table 1, by weight, by 99 parts of benzyl methacrylate, triethylene glycol dimethacrylate
After the mixing of 1 part of ester, and 1 part of initiator ABVN, according to 9：After 1 prepares, carried out under certain flow velocity with nitrogen
Advertise 30 minutes, the resin monomer that will be mixed is stored in standby in -30 DEG C of refrigerator；
(3) solidify：The brain piece that will be sealed is placed in baking oven in vacuum drying oven, and it is carried out according to the condition of cure of setting
Solidification, imaging is carried out by it is slowly dropped to after room temperature cooling, wherein condition of cure and is concretely comprised the following steps：Triggering temperature
Solidify at 40 DEG C of degree 8 hours, then solidify 12 hours at 45 DEG C of initiation temperature, then solidification 6 is small at 50 DEG C of initiation temperature
When, finally solidify 3 hours at 55 DEG C of initiation temperature.
Comparative example 6
The commercial embedding medium (resin monomer, crosslinking agent and initiator) of comparative example use, and present invention use
Biological tissue's transparence effect is compared after embedding medium embedding.
A kind of embedding method of the embedding medium of biological tissue, comprises the following steps：
Step (1) and step (2) are with embodiment 1.
(3) solidify：Embedding medium is as shown in table 1, by weight, by the monomers 90 of acrylate monomer HM 20 shown in table 1
Part and 10 parts of 20 crosslinking agents of crosslinking agent HM mixture and and 1 part of initiator azodiisobutyronitrile compare 99 according to weight:1 mixing
Afterwards, carried out under certain flow velocity advertising 30 minutes with nitrogen, the resin monomer that will be mixed is stored in standby in -30 DEG C of refrigerator
With；
After the spare resin monomer that the mouse brain that will finally permeate is placed in capsule and addition is mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, imaging is carried out by it is slowly dropped to after room temperature cooling, wherein solid
Change condition and concretely comprise the following steps：Solidify 8 hours at 40 DEG C of initiation temperature, then solidify 12 hours at 45 DEG C of initiation temperature,
Then solidify 6 hours at 50 DEG C of initiation temperature, finally solidify 3 hours at 55 DEG C of initiation temperature.
Table 1
Table 1 is embodiment 1-5 and the species of the embedding medium of the use of comparative example 6, proportioning and refraction after embedding medium allotment
Rate list.
Above-described embodiment 1~5 and sample obtained in comparative example 6 are tested by the following method：
Fluorescence microscope test after embedding mouse brain：
Sample：The mouse brain of transgenic mice or SABC is embedded into diameter 1cm according to above-mentioned flow, it is high
1.5cm cylinders, are imaged after being cut flat with using diamond tool.Instrument：The double femto-second laser multiphoton microscopes of German Zeiss.
Test condition：Depending on sample concrete condition.
Fig. 2 is the photo that embodiment 1 embeds biological tissue, biological tissue's fully transparentization, it can be seen that on background paper
Black grid.
Fig. 3 is that embodiment 2 embeds front and rear biological tissue's Intrinsic fluorescence Protein G FP fluorescence intensities contrast, the GFP after embedding
Fluorescence Increasing, the ratio before and after embedding is probably 126%, it can be seen that this transparent resin can be very good to keep endogenous
Volume contraction causes fluorescence molecule to be assembled after fluorescin, and dehydration, so as to improve fluorescence intensity.
Fig. 4 is that embodiment 3 embeds the front and rear fluorescence intensities of biological tissue's extrinsic fluorescence dyestuff Alexa 488 contrast；Can be with
Find out that fluorescence intensity is 80% or so of reset condition, this this transparent resin of explanation can meet extrinsic fluorescence dyestuff
Embedding.
Fig. 5 is the 3D neural network structures of the endogenous GFP marks biological tissue of the embedding of embodiment 4；Can be with neuron
Fine structure is all kept well, can carry out the embedding of substantially product sample.
Fig. 6 is that embodiment 5 embeds front and rear biological tissue's extrinsic fluorescence dyestuff CY3 fluorescence intensities contrast；It can be seen that glimmering
Luminous intensity is 219% or so of reset condition, because dehydration after-contraction causes fluorescence molecule to be assembled, this this transparence tree of explanation
Fat can meet the embedding of extrinsic fluorescence dyestuff.
Fig. 7 is the photo that comparative example 6 embeds front and rear biological tissue, as seen from Figure 7, common acrylate bag
The biological tissue buried is substantially opaque, the refractive index and the folding of biological tissue of resin after the common acrylate cures of this explanation
Penetrate rate mismatch.
As it will be easily appreciated by one skilled in the art that the foregoing is only presently preferred embodiments of the present invention, it is not used to
The limitation present invention, all any modification, equivalent and improvement made within the spirit and principles in the present invention etc., all should include
Within protection scope of the present invention.
Claims (10)
1. a kind of embedding medium of smooth transparence biological tissue, it is characterised in that the embedding medium is wrapped to the biological tissue
Solidified resin is formed after burying, the solidified resin has the refractive index for matching with the biological tissue, so that the life
Thing tissue realizes light transparence.
2. embedding medium as claimed in claim 1, it is characterised in that the refractive index of the monomer of the embedding medium is 1.46~1.55
Between, preferably 1.48~1.53.
3. embedding medium as claimed in claim 1, it is characterised in that the embedding medium is resin embedding agent, the resin embedding
Agent is preferably acrylic resin.
4. embedding medium as claimed in claim 3, it is characterised in that the acrylic resin includes two components of A, B, the A
Component by weight, including 90 to 100 parts of acrylate resin monomer and 0 to 10 part of diacrylate crosslinking agents；Institute
State B component by weight, including the alkenyl polymerization initiator less equal than 10 parts；The weight of the component A and B component is matched somebody with somebody
Than 90:10 to 999:Between 1.
5. embedding medium as claimed in claim 4, it is characterised in that the acrylate resin monomer contains phenyl for side base
Allyl compound, preferably 2- phenoxyethyl acrylates, phenyl methacrylate, benzyl acrylate, methacrylic acid
One or more in benzyl ester and/or phenyl acrylate, more preferably 2- phenoxyethyl acrylates and/or methyl-prop
Olefin(e) acid phenyl ester.
6. embedding medium as claimed in claim 4, it is characterised in that the diacrylate acid esters crosslinking agent is to contain diene base
Acrylate compounds, preferably ethylene glycol dimethacrylate, triethylene glycol diacrylate, the double acrylic acid of diethylene glycol
In ester, TEG dimethylacrylate, tetraethylene glycol diacrylate and/or TEGDMA one or
It is various, more preferably tetraethylene glycol diacrylate and/or TEGDMA.
7. embedding medium as claimed in claim 4, it is characterised in that the alkenyl polymerization initiator be azo-initiator and/
Or peroxide initiator, the azo-initiator is azodiisobutyronitrile, tert-butyl azodicarboxylate, ABVN
And/or one or more of azo-bis-iso-dimethyl, preferably azodiisobutyronitrile and/or ABVN；It is described
Peroxide initiator is dibenzoyl peroxide, peroxidating double lauroyl, di-t-butyl peroxide, cumyl peroxide peroxides
Change t-butyl perbenzoate, peroxidating trimethylacetic acid tertiary butyl ester di-isopropyl peroxydicarbonate and/or the ring of peroxy dicarbonate two
The double lauroyl of one or more of own ester, preferably dibenzoyl peroxide and/or peroxidating.
8. a kind of embedding method of smooth transparence biological tissue, it is characterised in that comprise the following steps：
(1) biological tissue's pretreatment：Biological tissue is fixed and dewater treatment, pretreated biological tissue's sample is obtained
This；
(2) permeate：By the pretreated biological tissue obtained in step (1) be immersed at infiltration temperature and light protected environment as
In embedding medium described in claim 1~7 any one, the embedding medium is set fully to permeate biological tissue, after being permeated
Biological tissue；The infiltration temperature is preferably -10 DEG C to 4 DEG C；
(3) solidify：Biological tissue after the infiltration that will be obtained in step (2) is solidified according to the heating schedule of setting.
9. embedding method as claimed in claim 8, it is characterised in that step (3) heating schedule for setting as：Triggering
Temperature is initiation solidification at 40 DEG C to 55 DEG C, and hardening time is 12 hours to 36 hours；The heating schedule of the setting is consolidated
Change specific steps to be preferably：Solidify 8 hours at 40 DEG C of initiation temperature, then solidify 12 hours at 45 DEG C of initiation temperature, so
Solidify 6 hours at 50 DEG C of initiation temperature afterwards, finally solidify 3 hours at 55 DEG C of initiation temperature.
10. a kind of application of light transparence biological tissue embedding agent as described in claim 1~7 any one, its feature exists
In being applied to the optical imagery of biological tissue.
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