CN106866876B - A kind of embedding medium, embedding method and the application of smooth transparence biological tissue - Google Patents
A kind of embedding medium, embedding method and the application of smooth transparence biological tissue Download PDFInfo
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- CN106866876B CN106866876B CN201710209321.6A CN201710209321A CN106866876B CN 106866876 B CN106866876 B CN 106866876B CN 201710209321 A CN201710209321 A CN 201710209321A CN 106866876 B CN106866876 B CN 106866876B
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- biological tissue
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- 229920001223 polyethylene glycol Polymers 0.000 description 1
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- 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
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- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- UWHCKJMYHZGTIT-UHFFFAOYSA-N tetraethylene glycol Chemical compound OCCOCCOCCOCCO UWHCKJMYHZGTIT-UHFFFAOYSA-N 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Materials For Medical Uses (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses the embedding mediums and its embedding method of a kind of smooth transparence biological tissue.Embedding medium is high refractive index acrylate, including two components of A, B；Component A includes 90 to 100 parts of acrylate resin monomer, 0 to 10 part of diacrylate crosslinking agents by weight；B component includes the alkenyl polymerization initiator less equal than 10 parts by weight；The weight proportion of component A and B component is in 90:10 between 999:1.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, it is high to embed success rate, 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 fields, embedding medium, embedding more particularly, to a kind of smooth transparence biological tissue
Method and application.
Background technique
The network structure for studying cranial nerve has the mental disease of the treatment mankind and cognition and emotion for studying the mankind etc.
It is of great importance, obtains the 3D neural network structure of full brain range and the resolution ratio for reaching cynapse rank is one for fluorescence imaging
A cross-cutting huge challenge, while can also promote the technological progress of the following entire sciemtifec and technical sphere, especially from complete system
The relationship between brain structure and function is studied in function of organization.It realizes and studies biological tissue in complete biosystem
Individual molecule structural information still remains very big difficulty.When carrying out optical imagery for the biological tissue that fluorescence probe marks,
The mainstream technology to large sample imaging external at present is to lead to biological tissue's transparence combination mating plate fluorescence microscope imaging technique
Crossing will realize that bilateral light excitation fluorescent molecule realizes successively imaging after biological tissue's transparence, this method image taking speed is fast, but
It is many kinds of of currently used liquid reagent transparence sample making technology, respectively there are advantage and disadvantage.
Refraction coefficient (RIs) of the light between different substances is different, and is caused scattered power of the light in system and is increased
Greatly.It is exactly that imageable target is made to reach the smallest light scattering effect that light is transparent, and lipid is cause mammal brain light to scatter main
Factor.Therefore, can be realized by removing the lipid on cell membrane and matching the refraction coefficient of lipid surrounding biological tissue by biological group
It knits transparent.Existing biological tissue's transparence technology is divided into chemical method and physics and two kinds of means of chemical bonding.Using chemistry
The method of reagent can be divided into aqueous transparent reagent and oiliness transparence reagent again.Aqueous transparent reagent saves fluorescin
Effect it is preferable, it is more mild to the processing of biological tissue, but the transparence rate of aqueous transparent reagent is lower, and transparence
The size of biological tissue easily expands afterwards, influences the analysis to biological tissue's primary morphology.Oiliness transparence reagent it is transparent
Change rate is very fast, and the change in size of transparence degree height and 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 remove lipoprotein function to biological tissue and realize transparence under the action of electric field, the operating process of this method
Complexity, repeatability is poor, and the requirement to equipment and personnel is high.Meanwhile liquid light transparent agent has certain toxicity more, prepares sample
This program is various, and specimen storage is more difficult and its fluorescence signal is difficult to keep for a long time.The transparent metaplasia of the liquid reagent of the prior art
The reagent and transparence method of object tissue more or less have a degree of destruction to the Intrinsic fluorescence albumen of biological tissue,
Fluorescence cannot well be kept.
In addition, only relying on biological tissue's endogenous fluorescence albumen in order to meet cranial nerve network structure imaging and tending not to completely
The requirement of sufficient fluorescence imaging realizes resin embedding by using external source fluorescent dye immunohistochemical markers simultaneously, enhances fluorescence, from
And increase imaging effect, but the embedding medium that the resin embedding technology of the prior art uses tends not to guarantee external source fluorescent dye
Molecular fluorescence keeps good, and therefore, needing one kind, to be able to maintain endogenous fluorescence effect good, can be compatible with external source fluorescent dye mark
Note realizes that specimen storage is simple and can keep biological tissue's transparence technology of fluorescence signal and simple process for a long time.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of packets of smooth transparence biological tissue
Agent and methods and applications are buried, its object is to have the embedding medium of appropriate index by selection, 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 the chemical reagent and the transparence side that well thus solve existing liquid reagent transparence biological tissue
Compatible, specimen storage inconvenience is difficult to there are fluorescent protein labeling and fluorochrome label in method and fluorescence signal is difficult to keep for a long time
The technical issues of.
To achieve the above object, according to one aspect of the present invention, a kind of embedding of smooth transparence biological tissue is provided
Agent, the embedding medium form solidified resin, the solidified resin and the biological group after being embedded to the biological tissue
It knits with the refractive index to match, so that the biological tissue be made to realize 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 in 90:10 between 999:1.
Preferably, the acrylate resin monomer is the allyl compound that side group contains phenyl.
Preferably, the acrylate resin monomer is 2- phenoxyethyl acrylate, phenyl methacrylate, propylene
One of acid benzyl ester, benzyl methacrylate and/or phenyl acrylate are a variety of.
Preferably, the acrylate resin monomer is 2- phenoxyethyl acrylate 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, tetraethylene glycol 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, azobisisoheptonitrile and/or azo-bis-iso-dimethyl
Kind is a variety of.
Preferably, the alkenyl polymerization initiator is azodiisobutyronitrile and/or azobisisoheptonitrile；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 a variety of.
Preferably, the peroxide initiator is dibenzoyl peroxide and/or the double lauroyl of peroxidating.
Other side according to the invention provides a kind of embedding method of smooth transparence biological tissue, including following
Step:
(1) biological tissue pre-processes: biological tissue being fixed and dehydration, pretreated biological tissue is obtained
Sample；
(2) it permeates: the pretreated biological tissue obtained in step (1) is impregnated at infiltration temperature and light protected environment
In the embedding medium, make the fully penetrated biological tissue of the embedding medium, the biological tissue after being permeated；The infiltration temperature
Preferably -10 DEG C to 4 DEG C of degree；
(3) solidify: the biological tissue after the infiltration obtained in step (2) is solidified according to the temperature program of setting.
Preferably, the temperature program of step (3) described setting are as follows: cause solidification at being 40 DEG C to 55 DEG C in initiation temperature,
Curing time is 12 hours to 36 hours.
Preferably, the temperature program of the setting carries out solidification specific steps are as follows: it is small to solidify 8 at 40 DEG C of initiation temperature
When, then solidify at 45 DEG C of initiation temperature 12 hours, then solidifies at 50 DEG C of initiation temperature 6 hours, finally causing temperature
Solidify 3 hours at 55 DEG C of degree.
Other side according to the invention provides the application of light transparence biological tissue embedding agent described in one kind,
Optical imagery applied to biological tissue.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect.
(1) biotissue optical clearing method provided by the invention uses high refractive index acrylate embedding medium, by solid
Resin and the index matching of biological tissue realize the fast transparent of biological tissue after change；
(2) reagent that transparentization of the invention is simple, uses is cheap and easily-available；
(3) method of smooth transparence biological tissue provided by the invention, can be adapted for mating plate fluorescence microscope and machinery
Chromatography combines wide field fluorescence microscope, can also potentially be applied to Electronic Speculum.
(4) method of smooth transparence biological tissue provided by the invention is suitable for Intrinsic fluorescence albumen and extrinsic fluorescence
The transparence of dye marker biological tissue is kept for the fluorescence signal time long, the fast imaging of biological tissue may be implemented.
Detailed description of the invention
Fig. 1 is the process 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 the biological tissue's Intrinsic fluorescence Protein G FP fluorescence intensity comparison of the embedding of embodiment 2 front and back；
Fig. 4 is 488 fluorescence intensity of the biological tissue extrinsic fluorescence dyestuff Alexa comparison of the embedding of embodiment 3 front and back；
Fig. 5 is the 3D neural network structure for the endogenous GFP label biological tissue that embodiment 4 embeds；
Fig. 6 is the biological tissue's extrinsic fluorescence dyestuff CY3 fluorescence intensity comparison of the embedding of embodiment 5 front and back；
Fig. 7 is the photo that comparative example 6 embeds biological tissue.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
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 in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
The embedding medium of smooth transparence biological tissue proposed by the present invention, embedding medium are resin embedding agent, for example are acrylic acid
Resin, acrylic resin include two components of A, B:
Component A by weight, the diacrylate including 90 to 100 parts of acrylate resin monomer and 0 to 10 part
Crosslinking agent；
B component by weight, including being less equal than 10 parts of alkenyl polymerization initiator；
The weight proportion of component A and B component is in 90:10 between 999:1.
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, in this way, working as
After the embedding medium embeds biological tissue, solidified resin and the biological tissue formed after embedding medium monomer polymerization has phase
Matched refractive index, can be achieved with the light transparence of biological tissue in this way, 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 group, for example is 2- Phenoxyethyl
One of acrylate, phenyl methacrylate, benzyl acrylate and/or phenyl acrylate are a variety of, preferably 2- benzene oxygen
Base ethyl propylene acid esters and/or phenyl methacrylate.This side group contains the acrylate monomer folding with higher of phenyl
Rate is penetrated, the refractive index of resin monomer can be improved on the whole, the refractive index after resin monomer polymerization can further increase.
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, tetraethylene glycol dimethylacrylate, tetraethylene glycol
One or more in diacrylate and/or triethylene glycol dimethacrylate, preferably tetraethylene glycol diacrylate and/or
Triethylene glycol dimethacrylate.It is this to contain double propenyl ester compound as crosslinking agent and solidified resin be made to be formed
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
Bis-isobutyronitrile, tert-butyl azodicarboxylate, azobisisoheptonitrile and/or azo-bis-iso-dimethyl it is one or more,
Preferably azodiisobutyronitrile and/or azobisisoheptonitrile；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 two diisopropyl carbonates and/or di-cyclohexylperoxy di-carbonate be one or more, preferably dibenzoyl peroxide and/
Or the double lauroyl of peroxidating.Polymerization temperature and fluorescence intensity when the selection of initiator affects embedding, packet provided by the invention
Burying agent can cause in middle low temperature, guarantee fluorescin activity to guarantee fluorescence intensity.
The embedding of biological tissue is carried out using above-mentioned embedding medium and realizes the light transparence method of biological tissue, including is following
Step:
(1) biological tissue pre-processes: biological tissue being fixed and dehydration, pretreated biological tissue is obtained
Sample；
(2) it permeates: by the pretreated biological tissue obtained in step (1) in certain infiltration temperature and light protected environment
Under be immersed in the embedding medium as described in claim 1~7 any one, make the fully penetrated biological tissue of the embedding medium, obtain
Biological tissue after must permeating；
(3) solidify: the biological tissue after the infiltration obtained in step (2) is solidified according to the temperature program of setting.
Wherein, step (2) the infiltration temperature is -10 DEG C to 4 DEG C.
The temperature program of step (3) described setting are as follows: in initiation temperature be that 40 DEG C to 55 DEG C initiation solidify, curing time is
12 hours to 36 hours；The temperature program of setting carries out solidification specific steps are as follows: solidifies 8 hours at 40 DEG C of initiation temperature, so
Solidify at 45 DEG C of initiation temperature afterwards 12 hours, then solidifies 6 hours at 50 DEG C of initiation temperature, finally at 55 DEG C of initiation temperature
Lower solidification 3 hours.
The embedding medium and method of smooth transparence biological tissue provided by the invention mainly use the acrylic acid of high refractive index
Its refractive index further increases after polyisocyanate polyaddition, when the refractive index 1.56 with dewatered biological tissue matches, biological tissue
It will realize transparence.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, transparence cannot be carried out to biological tissue.The present invention passes through the polymerized monomer of selection appropriate index, crosslinking agent
With initiator, deploy embedding medium in component A and B component composition proportion, deploy monomer refractive index be allowed to polymerization after just with
The index matching of dewatered biological tissue uses refractive index for 1.50 or so liquid methacrylate monomer, aggregates into solid
Refractive index after state resin is 1.56 or so.The higher refractive index of this monomer is to lead to because side group contains phenyl ring to improve light
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, suitable for the mating plate microscope of biological tissue's piece and large sample at
Picture, while applying also for the large sample automation successively imaging of automatic cutting system and fluorescence microscope joint technology, can also be with
Potentially it is applied to Electronic Speculum to be imaged.
The reaction principle that high refractive index monomers embed biological tissue is as follows:
The embedding medium that light transparence of the present invention biological tissue uses is the embedding medium of response type crosslinked bio tissue, using this
The embedding medium of response type crosslinked bio tissue, autofluorescence background is low, light transmittance is high, embedding process is simple, resin shrinkage rate
It is low and the advantages of can preferably keep neural fine structure, have both epoxy resin and acrylate, it is applicable in biological tissue
Plasticity embedding, success rate is high, and imaging is cut in the embedding especially suitable for substantially product sample and the full brain of mouse brain.
The following are embodiments:
Examples 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 resin embedding biology
The process of transparency of organization method, as shown in Figure 1, comprising the following steps:
(1) biological tissue pre-processes: being placed in 4% paraformaldehyde (PFA) rear fix firstly, will take out after mouse brain perfusion
24 hours；Then it is 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 ,+95 tetrahydrofuran/2 hour+100% of+75% tetrahydrofuran/2 hour of 50% tetrahydrofuran/2 hour
+ 100% tetrahydrofuran/4 hour of tetrahydrofuran/2 hour；
(2) it permeates: and then be immersed in dewatered mouse brain in the resin monomer got ready under 4 DEG C of light protected environments 2 hours
Liquid is changed again, is then impregnated again 2 days；
(3) solidify: embedding medium is as shown in table 1, by weight, by acrylate monomer 2- phenoxy group second shown in table 1
90 parts of base acrylate and 10 parts of crosslinking agent triethylene glycol dimethacrylate of mixture and and two isobutyl of initiator azo
After 10 parts of nitrile according to weight ratio 99:1 mixing, carried out advertising 30 minutes under certain flow velocity with nitrogen, the resin that will be mixed
Monomer storage is spare in -30 DEG C of refrigerator；
Finally the mouse brain permeated is placed in capsule and is added after the spare resin monomer mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, is slowly dropped to that imaging can be carried out after room temperature is cooling after it, wherein solid
Change condition and specific steps are as follows: solidify at 40 DEG C of initiation temperature 8 hours, then solidifies 12 hours at 45 DEG C of initiation temperature,
Then solidify at 50 DEG C of initiation temperature 6 hours, 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, includes the following steps:
Step (1) and step (2) are the same as embodiment 1.
(3) solidify: embedding medium is as shown in table 1, by weight, by acrylate monomer methacrylic acid shown in table 1
95 parts of phenyl ester and 5 parts of crosslinking agent tetraethylene glycol diacrylate of mixture with and 1 part of initiator azobisisoheptonitrile according to weight
After 99:1 mixing, is carried out advertising 30 minutes under certain flow velocity with nitrogen, the resin monomer mixed is stored in -30 DEG C
Refrigerator in it is spare；
Finally the mouse brain permeated is placed in capsule and is added after the spare resin monomer mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, is slowly dropped to that imaging can be carried out after room temperature is cooling after it, wherein solid
Change condition and specific steps are as follows: solidify at 40 DEG C of initiation temperature 8 hours, then solidifies 12 hours at 45 DEG C of initiation temperature,
Then solidify at 50 DEG C of initiation temperature 6 hours, finally solidify 3 hours at 55 DEG C of initiation temperature.
Embodiment 3
(1) biological tissue pre-processes: being placed in 4% paraformaldehyde (PFA) rear fix firstly, will take out after mouse brain perfusion
24 hours；Mouse brain is subjected to bobbing machine again and carries out slicing treatment, is then rinsed 3 times, every time 8 hours using PBS solution.It will fix
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 to 100 microns of murine brain pieces carry out punching processing 12 hours, then plus
The serum for entering 10% is closed 12 hours, and primary antibody is added at 37 DEG C according still further to the ratio of 500:1 after rinsing 1 hour/3 times using PBS
It is protected from light lower vibration to be incubated for 2 days, 37 DEG C of ratios being protected from light after lower vibration is incubated for 2 days according still further to 800:1 are added 4 DEG C of secondary antibody and are protected from light lower vibration
Dynamic incubation is rinsed 1 hour/3 times after 8 hours using PBS.
(2) it permeates: 100% tetrahydrofuran/10 minute of brain piece after fluorescent dye immunohistochemistry；It then will be dewatered
Brain piece, which is placed in resin monomer, to permeate 10 minutes, is placed on glass slide, after dripping upper 2 drop resin monomer, covered；Wherein set
Alicyclic monomer are as follows: according to the proportion in table 1, by weight, and by 95 parts of phenyl methacrylate, triethylene glycol dimethacrylate
After 5 parts of ester mixing, and 5 parts of initiator dilauroyl peroxide, after being prepared according to 995:5, with nitrogen under certain flow velocity
Advertise 30 minutes, the resin monomer mixed is stored in spare in -30 DEG C of refrigerator；
(3) solidify: the brain piece sealed is placed in baking oven in vacuum drying oven, it is carried out according to the condition of cure of setting
Solidification is slowly dropped to that imaging can be carried out after room temperature is cooling after it, wherein condition of cure and specific steps are as follows: causing temperature
Solidify 8 hours at 40 DEG C of degree, then solidifies at 45 DEG C of initiation temperature 12 hours, 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, includes the following steps:
Step (1) and step (2) are the same as embodiment 1.
(3) solidify: embedding medium is as shown in table 1, by weight, by acrylate monomer 2- phenoxy group second shown in table 1
After 100 parts of base acrylate mix with 10 parts of initiator benzoyl peroxide according to weight ratio 99:1, with nitrogen in certain stream
It carries out advertising 30 minutes under speed, the resin monomer mixed is stored in spare in -30 DEG C of refrigerator；
Finally the mouse brain permeated is placed in capsule and is added after the spare resin monomer mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, is slowly dropped to that imaging can be carried out after room temperature is cooling after it, wherein solid
Change condition and specific steps are as follows: solidify at 40 DEG C of initiation temperature 8 hours, then solidifies 12 hours at 45 DEG C of initiation temperature,
Then solidify at 50 DEG C of initiation temperature 6 hours, finally solidify 3 hours at 55 DEG C of initiation temperature.
Embodiment 5:
(1) biological tissue pre-processes: being placed in 4% paraformaldehyde (PFA) rear fix firstly, will take out after mouse brain perfusion
24 hours；Mouse brain is subjected to bobbing machine again and carries out slicing treatment, is then rinsed 3 times, every time 8 hours using PBS solution.It will fix
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 processing 12 hours to 100 microns of murine brain pieces, is then added 10%
Serum close 12 hours, using PBS rinse 1 hour/3 times after according still further to 500:1 ratio be added primary antibody in the case where 37 DEG C are protected from light
Vibration is incubated for 2 days, and 37 DEG C of 4 DEG C of secondary antibody of ratios additions being protected from light after lower vibration is incubated for 2 days according still further to 800:1 are protected from light lower vibration and are incubated for
It is rinsed 1 hour/3 times after 8 hours using PBS.
(2) it permeates: 100% tetrahydrofuran/10 minute of brain piece after fluorescent dye immunohistochemistry；It then will be dewatered
Brain piece, which is placed in resin monomer, to permeate 10 minutes, is placed on glass slide, after dripping upper 2 drop resin monomer, covered；Wherein set
Alicyclic monomer are as follows: according to the proportion in table 1, by weight, and by 99 parts of benzyl methacrylate, triethylene glycol dimethacrylate
After 1 part of ester mixing, and 1 part of initiator azobisisoheptonitrile, after being prepared according to 9:1, carried out under certain flow velocity with nitrogen
It advertises 30 minutes, the resin monomer mixed is stored in spare in -30 DEG C of refrigerator；
(3) solidify: the brain piece sealed is placed in baking oven in vacuum drying oven, it is carried out according to the condition of cure of setting
Solidification is slowly dropped to that imaging can be carried out after room temperature is cooling after it, wherein condition of cure and specific steps are as follows: causing temperature
Solidify 8 hours at 40 DEG C of degree, then solidifies at 45 DEG C of initiation temperature 12 hours, 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 comparative example is using commercial embedding medium (resin monomer, crosslinking agent and initiator), come what is used with the present invention
Biological tissue's transparence effect is compared after embedding medium embeds.
A kind of embedding method of the embedding medium of biological tissue, includes the following steps:
Step (1) and step (2) are the same as embodiment 1.
(3) solidify: embedding medium is as shown in table 1, by weight, by 20 monomer 90 of acrylate monomer HM shown in table 1
Part and 10 parts of 20 crosslinking agent of crosslinking agent HM mixture with and 1 part of initiator azodiisobutyronitrile mixed according to weight ratio 99:1
Afterwards, it is carried out advertising 30 minutes under certain flow velocity with nitrogen, the resin monomer mixed is stored in standby in -30 DEG C of refrigerator
With；
Finally the mouse brain permeated is placed in capsule and is added after the spare resin monomer mixed in vacuum drying oven,
It is solidified according to the condition of cure of setting, is slowly dropped to that imaging can be carried out after room temperature is cooling after it, wherein solid
Change condition and specific steps are as follows: solidify at 40 DEG C of initiation temperature 8 hours, then solidifies 12 hours at 45 DEG C of initiation temperature,
Then solidify at 50 DEG C of initiation temperature 6 hours, finally solidify 3 hours at 55 DEG C of initiation temperature.
Table 1
Type, proportion and the embedding medium for the embedding medium that table 1 is embodiment 1-5 and comparative example 6 uses reflect after deploying
Rate list.
Sample made from above-described embodiment 1~5 and comparative example 6 is tested by the following method:
Fluorescence microscope test after embedding mouse brain:
Sample: embedding into diameter 1cm for the mouse brain of transgenic mice either immunohistochemistry according to the above process, high
1.5cm cylinder is imaged after being cut flat with using diamond tool.Instrument: the German bis- femto-second laser multiphoton microscopes of 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 the biological tissue's Intrinsic fluorescence Protein G FP fluorescence intensity comparison of the embedding of embodiment 2 front and back, the GFP after embedding
Fluorescence enhancement, the ratio for embedding front and back is probably 126%, it can be seen that this transparent resin can be very good to keep endogenous
Fluorescin, and volume contraction causes fluorescent molecule to be assembled after dehydration, to improve fluorescence intensity.
Fig. 4 is 488 fluorescence intensity of the biological tissue extrinsic fluorescence dyestuff Alexa comparison of the embedding of embodiment 3 front and back；It can be with
Find out that fluorescence intensity is 80% of reset condition or so, this illustrates that this transparent resin can satisfy extrinsic fluorescence dyestuff
Embedding.
Fig. 5 is the 3D neural network structure for the endogenous GFP label biological tissue that embodiment 4 embeds；It can be with neuron
Fine structure is all kept well, can carry out the embedding of substantially product sample.
Fig. 6 is the biological tissue's extrinsic fluorescence dyestuff CY3 fluorescence intensity comparison of the embedding of embodiment 5 front and back；It can be seen that glimmering
Luminous intensity is 219% of reset condition or so, and since dehydration after-contraction causes fluorescent molecule to be assembled, this illustrates this transparence tree
Rouge can satisfy the embedding of extrinsic fluorescence dyestuff.
Fig. 7 is the photo of the embedding of comparative example 6 front and back biological tissue, as seen from Figure 7, common acrylate packet
The biological tissue buried is substantially opaque, this illustrates the refractive index of resin and the folding of biological tissue after common acrylate cures
Penetrate rate mismatch.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of embedding medium of smooth transparence biological tissue, which is characterized in that the embedding medium wraps the biological tissue
Solidified resin is formed after burying, the solidified resin and the biological tissue have the refractive index to match, to make the life
Object tissue realizes light transparence；
The embedding medium is acrylic resin；The acrylic resin includes two components of A, B, the component A by weight,
Diacrylate crosslinking agents including 90 to 100 parts of acrylate resin monomer and 0 to 10 part；The B component is by weight
Meter, the alkenyl polymerization initiator including being less equal than 10 parts；The weight proportion of the component A and B component is in 90:10 to 999:
Between 1；The acrylate resin monomer is the allyl compound that side group contains phenyl.
2. embedding medium as described in claim 1, which is characterized in that the refractive index of the monomer of the embedding medium is 1.46~1.55
Between.
3. embedding medium as described in claim 1, which is characterized in that the refractive index of the monomer of the embedding medium be 1.48~
1.53。
4. embedding medium as described in claim 1, which is characterized in that the acrylate resin monomer is 2- Phenoxyethyl third
One of olefin(e) acid ester, phenyl methacrylate, benzyl acrylate, benzyl methacrylate and/or phenyl acrylate are a variety of.
5. embedding medium as described in claim 1, which is characterized in that the diacrylate crosslinking agents are third containing diene base
Enoic acid ester compounds.
6. embedding medium as described in claim 1, which is characterized in that the alkenyl polymerization initiator be azo-initiator and/
Or peroxide initiator, the azo-initiator are azodiisobutyronitrile, tert-butyl azodicarboxylate, azobisisoheptonitrile
And/or azo-bis-iso-dimethyl is one or more；The peroxide initiator is dibenzoyl peroxide, peroxidating pair
Lauroyl, di-t-butyl peroxide, cumyl peroxide, peroxidized t-butyl perbenzoate, peroxidating trimethylacetic acid tert-butyl
Ester, di-isopropyl peroxydicarbonate and/or di-cyclohexylperoxy di-carbonate are one or more.
7. a kind of embedding method of smooth transparence biological tissue, which comprises the following steps:
(1) biological tissue pre-processes: biological tissue being fixed and dehydration, pretreated biological tissue's sample is obtained
This；
(2) permeate: the pretreated biological tissue obtained in step (1) is immersed at infiltration temperature and light protected environment as
In embedding medium described in claim 1~6 any one, make the fully penetrated biological tissue of the embedding medium, after being permeated
Biological tissue；The infiltration temperature is preferably -10 DEG C to 4 DEG C；
(3) solidify: the biological tissue after the infiltration obtained in step (2) is solidified according to the temperature program of setting.
8. embedding method as claimed in claim 7, which is characterized in that the temperature program of step (3) described setting are as follows: causing
Temperature is to cause solidification at 40 DEG C to 55 DEG C, and curing time is 12 hours to 36 hours.
9. embedding method as claimed in claim 7, which is characterized in that the temperature program of step (3) described setting is solidified
Specific steps are as follows: solidify at 40 DEG C of initiation temperature 8 hours, then solidify 12 hours at 45 DEG C of initiation temperature, then drawing
Solidify 6 hours under hair temperature 50 C, finally solidifies 3 hours at 55 DEG C of initiation temperature.
10. a kind of application of the light transparence biological tissue embedding agent as described in claim 1~6 any one, feature exist
In optical imagery applied to biological tissue.
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| CN110108537B (en) * | 2018-02-01 | 2020-08-25 | 中国科学技术大学 | Embedding medium and embedding method for embedding biological tissue |
| CN108680417B (en) * | 2018-04-20 | 2019-08-06 | 吴礼高 | A kind of preparation method of easy elution biological tissue embedding agent |
| CN109991056B (en) * | 2019-04-04 | 2021-12-14 | 深圳市通用氢能科技有限公司 | Formula and preparation method of epoxy resin embedding agent for low-infiltration fuel cell electron microscope slices |
| CN110243828B (en) * | 2019-07-18 | 2021-07-30 | 华中科技大学 | Biological tissue three-dimensional imaging method based on convolutional neural network |
| US20220357246A1 (en) * | 2019-08-07 | 2022-11-10 | JelloX Biotech Inc. | Composition and method for rendering biological material |
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| US3489712A (en) * | 1964-07-15 | 1970-01-13 | Newton G Leveskis | Composition and method for mounting specimens on slides |
| CN1329245A (en) * | 2001-07-24 | 2002-01-02 | 上海市第六人民医院 | Biological tissue section embedding agent |
| CN106198170A (en) * | 2016-06-30 | 2016-12-07 | 华中科技大学 | A kind of biological tissue embedding agent and embedding method |
| CN106323708A (en) * | 2016-07-29 | 2017-01-11 | 浙江大学 | Transparent reagent, biological tissue transparency imaging method and application of transparent reagent |
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