CN109612811A - A kind of hydrogel embedding method for protecting mechanics of biological tissue and fluorescence - Google Patents
A kind of hydrogel embedding method for protecting mechanics of biological tissue and fluorescence Download PDFInfo
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- 238000003384 imaging method Methods 0.000 claims abstract description 9
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- 238000012719 thermal polymerization Methods 0.000 claims description 9
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- 239000002904 solvent Substances 0.000 claims description 6
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- 239000003513 alkali Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 3
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- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 2
- 235000019394 potassium persulphate Nutrition 0.000 claims description 2
- 239000000499 gel Substances 0.000 abstract description 10
- 150000003384 small molecules Chemical class 0.000 abstract description 3
- 230000000977 initiatory effect Effects 0.000 abstract description 2
- 210000004556 brain Anatomy 0.000 description 29
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 239000005090 green fluorescent protein Substances 0.000 description 12
- 210000005013 brain tissue Anatomy 0.000 description 10
- 239000008363 phosphate buffer Substances 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- 229930040373 Paraformaldehyde Natural products 0.000 description 7
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- 241000699670 Mus sp. Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000012466 permeate Substances 0.000 description 6
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
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- 230000010412 perfusion Effects 0.000 description 5
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- 230000008602 contraction Effects 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 241001529936 Murinae Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000012670 alkaline solution Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
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- 238000002955 isolation Methods 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 230000013011 mating Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- WJEIYVAPNMUNIU-UHFFFAOYSA-N [Na].OC(O)=O Chemical compound [Na].OC(O)=O WJEIYVAPNMUNIU-UHFFFAOYSA-N 0.000 description 1
- -1 acrylamido glycine amide Chemical compound 0.000 description 1
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000012937 correction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- G01N2001/305—Fixative compositions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention discloses a kind of hydrogel embedding method for protecting mechanics of biological tissue and fluorescence, comprising: pre-processes tissue, and prepares NAGA solution；Described pretreated group is woven in the NAGA solution and carries out infiltration processing；Group after infiltration is woven in NAGA solution and is polymerize；Biological tissue after polymerization is imaged in imaging system.Primary improvements of the invention are that the present invention provides a kind of small molecules to penetrate into biological tissue; in certain under conditions of initiation polymerization; form the three-dimensional net structure of tissue and gel molecular; protect mechanics of biological tissue and fluorescence; the method operation is succinct; it does not need to be carried out dehydrating biological tissue in use process, tissue deformation will not be caused, biological tissue's fluorescence can be saved for a long time.Tissue after embedding has a degree of transparent, can be used for the acquisition of the fine morphosis of biological tissue.
Description
Technical field
The invention belongs to biomedical engineering field more particularly to a kind of hydrogels for protecting mechanics of biological tissue and fluorescence
Embedding method.
Background technique
Fluorescent microscopic imaging is that we obtain the effective way of organism fluorescent marker information.It is glimmering in neuroscience field
Photoprotein is normally used for the completeness that transgenic technology and virus signature technically come tracer neural circuitry or labeled neurons
Research of state and some other biological question such as cell space counting etc..However existing a variety of fluorescent sample processing methods are but
It is all difficult to combine the fluorescence and morphosis of biological tissue.
It is long all to there is the sample process time in almost all of smooth clearing method, among sample not with sample surface transparency
The problems such as holding of consistent and fluorescence is bad, and biological sample can also be presented during transparency process different degrees of contraction or
Biological tissue's volume contraction to original 55%, CUBIC and PACT can be made biological tissue's volume expansion by expansion, UDISCO, together
When fluorescence also will appear certain be quenched.Although the fluorescent sample plasticity embedding method developed its can effectively save green
Fluorescent protein fluorescence, but it is poor to the preservation of red fluorescent protein fluorescence, and its sample process process is cumbersome, dehydration and resin polymerization
Link makes biological tissue's form certain deformation occur, although can the later period by image procossing carry out part correction,
It is time-consuming and laborious.
Summary of the invention
In view of this, the present invention provides a kind of biological tissue embedding method of micro-molecular hydrogel, the method operation is succinct,
It does not need to be carried out dehydrating biological tissue in use process, tissue deformation will not be caused, biological tissue can be saved for a long time
Fluorescence.Tissue after embedding has a degree of transparent, can be used for the acquisition of the fine morphosis of biological tissue.
In order to solve the above technical problems, the technical scheme is that penetrating into biological tissue using a kind of small molecule,
Cause polymerization under conditions of certain, form the three-dimensional net structure of tissue and gel molecular, protects mechanics of biological tissue and glimmering
Light.This method specifically includes:
Tissue is pre-processed, and prepares NAGA solution；
NAGA monomer and initiator, solvent are mixed to get NAGA solution；
Described pretreated group is woven in the NAGA solution and carries out infiltration processing；
Group after infiltration is woven in NAGA solution and is polymerize；
It will be imaged in biological tissue's reimaging system after polymerization.
It is preferably, described to pre-process tissue specifically:
Biological tissue is provided；
It is fixed after the biological tissue is carried out PFA, PBS rinsing.
Preferably, the solvent is selected from the aqueous solution or pure water of salt, acid or alkali.
Such as aqueous solution may include: distilled water+salt (phosphate etc.)=phosphate buffer；Distilled water+alkali (carbonic acid
Sodium, sodium hydroxide etc.)=alkaline solution；Distilled water+acid (hydrochloric acid etc.)=acid solution.
Preferably, the mass percent of the NAGA is 5%-50%.
Preferably, the initiator is selected from ammonium persulfate, potassium peroxydisulfate, azo diisopropyl fourth miaow hydrochloride (VA-05)
Or azo dicyclohexyl formonitrile HCN hydrochloride (VA-044).
Preferably, it is 0.01 ‰ -5 ‰ that the initiator, which accounts for the mass percent of system,.
Preferably, the group after infiltration is woven in NAGA solution and is polymerize specifically:
Tissue after infiltration is immersed in new NAGA solution, thermal polymerization in an oven.
Preferably, for the temperature of the thermal polymerization at 25-50 DEG C, the thermal polymerization time is 3-24h.
Preferably, the tissue after polymerization is imaged on an imaging device specifically:
Tissue after polymerization is placed in wide field fluorescence microscope, is imaged on Laser Scanning Confocal Microscope or Two Photon Fluorescence.
Primary improvements of the invention are that the present invention provides a kind of small molecules to penetrate into biological tissue, in certain item
Cause polymerization under part, form the three-dimensional net structure of tissue and gel molecular, protects mechanics of biological tissue and fluorescence, the method behaviour
Make succinctly, not needing to be carried out dehydrating biological tissue in use process, tissue deformation will not be caused, life can be saved for a long time
Object histofluorescence.Tissue after embedding has a degree of transparent, can be used for the acquisition of the fine morphosis of biological tissue.
Detailed description of the invention
Fig. 1 is that hydrogel provided in an embodiment of the present invention embeds biological tissue's schematic diagram；
Fig. 2 is that GFP provided in an embodiment of the present invention (green fluorescent protein) labeled neurons hydrogel embeds front and back form
Comparison；
Fig. 3 is that lectin provided in an embodiment of the present invention marks imaging results after the embedding of mouse cerebrovascular hydrogel；
Fig. 4 is that GFP labeled neurons hydrogel provided in an embodiment of the present invention embeds front and back fluorescence intensity comparison；
Fig. 5 is that GFP labeled neurons hydrogel provided in an embodiment of the present invention embeds front and back imaging depth comparison；
Fig. 6 is three-dimensional structure imaging results after multi-color marking murine brain hydrogel provided in an embodiment of the present invention embedding.
Specific embodiment
In order to make those skilled in the art more fully understand technical solution of the present invention, With reference to embodiment
The present invention is described in further detail.
The present invention uses NAGA (acrylamido glycine amide) molecule infiltration is solidifying into formation biological tissue-, biological tissue
A kind of biological tissue embedding method of hydrogel of glue three-dimensional network, this method specifically include:
Pre-treatment step pre-processes tissue, and prepares NAGA hydrogel solution；
Described pretreated group is woven in the NAGA solution and carries out infiltration processing by osmosis treatment step；
Polymerization embedding step, the group after infiltration is woven in NAGA solution and is polymerize；
Image-forming step will be imaged in biological tissue's reimaging system after polymerization.
Concrete principle is as shown in Figure 1, there are two in gel molecular NAGA and biological tissue (for mouse brain) embedding process
Step, 1, the crosslinking of the biomolecule such as amido bond and albumen of gel molecular；2, gel molecular polymerize to form three-dimensional network, by tissue
" package " wherein, forms gel molecular --- the three-dimensional net structure of biological tissue.NAGA hydrogel itself is due to double acryloyls
The presence that amine is built can form dihydrogen bond, and this dihydrogen bond structure makes gel molecular --- and the three-dimensional net structure of biological tissue is more
Add stabilization.
According to the present invention, by fixing after biological tissue is carried out PPA, PBS rinsing pre-processes biological tissue.
NAGA monomer and initiator, solvent are mixed to get NAGA solution.Wherein, the solvent is for distilled water or with distillation
The aqueous solution that water and other materials are made into；Such as aqueous solution can include:
Distilled water+salt (phosphate etc.)=phosphate buffer
Distilled water+alkali (sodium carbonate, sodium hydroxide etc.)=alkaline solution
Distilled water+acid (hydrochloric acid etc.)=acid solution.
The mass percent of NAGA described in the hydrogel is 5%-50%.The initiator is selected from ammonium persulfate, mistake
Potassium sulfate, azo diisopropyl fourth miaow hydrochloride (VA-05) or azo dicyclohexyl formonitrile HCN hydrochloride (VA-044).It is described to draw
The mass percent that hair agent accounts for system is 0.01 ‰ -5 ‰.
After pre-processing tissue, described group is woven in the NAGA solution and carries out infiltration processing, the time for permeating processing is
1min~3day, specific time of penetration can be adjusted by the difference of tissue, the tissue after infiltration be immersed in new
In NAGA solution, thermal polymerization in an oven.For the temperature of the thermal polymerization at 25-50 DEG C, the thermal polymerization time is 3-24h.
Tissue after polymerization is placed in wide field fluorescence microscope, is imaged on Laser Scanning Confocal Microscope or Two Photon Fluorescence.
Method provided by the invention is for more traditional resin embedding, paraffin embedding, and that eliminates dehydrating agents
The gradient penetration link of serial dehydration link and resin or paraffin only need to permeate the biological tissue after rinsing in NAGA solution
It is completely polymerizable embedding, the embedding of entire mouse brain also only need to time less than 2 days.
Inventors have found that the dehydration of object tissue dewatering agent and the infiltration of other organic reagents will lead to biological tissue's volume receipts
Contracting, due to inconsistent with the existence and content of water in the various structures of biological tissue, and many biological tissues such as brain all exists
Ventricles of the brain gap, therefore this contraction majority is nonlinear shrinkage.The preparation of hydrogel is originally while the hydrogel rich in moisture
Property determines that it relies on the presence of water environment and exists, therefore biological tissue does not need to be taken off during hydrogel embeds
Water process.Therefore it will not cause tissue deformation in entire embedding process.
There are two steps in hydrogel embedding process, 1, the crosslinking of the biomolecule such as amido bond and albumen of gel molecular；
2, polymerization occurs under the initiation of initiator and forms three-dimensional network for gel molecular, wherein by tissue " package ", is formed to knot of tissue
The protection of structure, therefore histofluorescence can be saved for a long time and maintain the fine morphosis of tissue.The folding of NAGA solution itself
Penetrating rate is 1.39, closer to the refractive index of lipid in tissue etc..When NAGA solution penetrates into biological tissue, in replacement tissue
Water can make the transparency of tissue increase.
The embedding copolymerization of 1 Green Fluorescent Protein mouse brain slice sample hydrogel of embodiment is burnt
10% urethane and 2% is injected intraperitoneally in Thy1-GFP (THY1 transgenosis) male mice at two monthly ages in step 1
Chloraldurate mixture is anaesthetized, and perfusion takes brain, and PFA (paraformaldehyde) fixes PBS (phosphate buffer) rinsing for 24 hours afterwards
24h；
Step 2, by Mouse Whole Brain in being sliced 100-500um on Lycra business vibratome；
Step 3 prepares NAGA solution, and wherein initiator selects VA-044；
The brain piece cut is put into NAGA solution and permeates 1-60min by step 4；
Step 5, the brain piece mounting that will have been permeated, slide surrounding are coated nail polish isolation air, and are polymerize in baking oven；
Step 6, by the brain piece after polymerization in being imaged on Laser Scanning Confocal Microscope.
By result shown in Fig. 2 find out hydrogel embedding after histofluorescence brightness be increased slightly, and neuron morphology without
Significant change illustrates that hydrogel embedding can be maintenance mechanics of biological tissue.
Embodiment 2tomato-lectin mark mouse blood vessel mouse brain slice hydrogel embedding Laser Scanning Confocal Microscope at
Picture
Step 1, in the 75 μ L tomato lectin (tomato lectin of C57BL/6 male mice tail vein injection at two monthly ages
Element) (DyLight 594labeled Tomato Lectin, 1 μ g/ μ L, 10mM HEPES, 0.15M NaCl；Vector
Laboratories), 10% urethane is injected intraperitoneally after 20min and 2% chloraldurate mixture is anaesthetized, perfusion takes brain,
It is fixed after PFA for 24 hours, PBS is rinsed for 24 hours；
Step 2, by Mouse Whole Brain in being sliced 100-500um on Lycra business vibratome；
Step 3 prepares NAGA solution, and wherein initiator selects VA-044, and 4 DEG C of refrigerators save；
The brain piece cut is put into NAGA solution and permeates 1-60min by step 4；
Step 5, the brain piece mounting that will have been permeated, slide surrounding are coated nail polish isolation air, are polymerize in baking oven；
Step 6, by the brain piece after polymerization in being imaged on Laser Scanning Confocal Microscope.
It can be seen that hydrogel embedding saves the official jargon structure of blood vessel well by result shown in Fig. 3.
3 Green Fluorescent Protein mouse brain slice sample hydrogel of embodiment embeds two photon imaging
10% urethane and the mixing of 2% chloraldurate is injected intraperitoneally in the Thy1-GFP male mice at two monthly ages in step 1
Object is anaesthetized, and perfusion takes brain, is fixed after PFA for 24 hours, PBS is rinsed for 24 hours；
Step 2 prepares NAGA solution, and wherein initiator selects VA-044, and 4 DEG C of refrigerators save；
Mouse Whole Brain is put into NAGA solution and permeates 0.5-3d by step 3；
The brain tissue permeated is put and fresh NAGA solution is added dropwise in test tube polymerize in baking oven by step 4；
Brain tissue after polymerization is repaired block by step 5, in being imaged on Two Photon Fluorescence.
Murine brain fluorescence imaging depth becomes larger after can be seen that hydrogel embedding by result shown in Fig. 4, further
Illustrate that hydrogel embedding tissues following MCAO in rats transparency increases.
4 Green Fluorescent Protein mouse brain slice sample hydrogel of embodiment embeds light-sheet imaging
10% urethane and the mixing of 2% chloraldurate is injected intraperitoneally in the Thy1-GFP male mice at two monthly ages in step 1
Object is anaesthetized, and perfusion takes brain, is fixed after PFA for 24 hours, PBS is rinsed for 24 hours；
Step 2, by Mouse Whole Brain in being sliced 1-5mm on Lycra business vibratome；
Step 3 prepares NAGA solution, and wherein initiator selects AIBI, and 4 DEG C of refrigerators save；
Half brain piece of mouse is put into NAGA solution and permeates 1-10h by step 4；
The brain tissue permeated is put and fresh NAGA solution is added dropwise in test tube polymerize in baking oven by step 5；
Brain tissue after polymerization is repaired block by step 6, in being imaged on Two Photon Fluorescence.
The brain tissue permeated is put and fresh NAGA solution is added dropwise in test tube polymerize in baking oven by step 7；
Brain tissue after polymerization is repaired block by step 8, is imaged on mating plate illumination microscope.
It can be seen that the biological tissue of hydrogel embedding is equally applicable to mating plate according to illumination by content shown in Fig. 5.
It is imaged after the embedding of 5 multi-color marking mouse brain hydrogel of embodiment
Step 1, after Thy1-GFP male mice tail vein injection 70ul the tomato lectin, 20min at two monthly ages
10% urethane is injected intraperitoneally and 2% chloraldurate mixture is anaesthetized, perfusion takes brain, and PBS rinsing for 24 hours is fixed after PFA
24h。
Step 2, by Mouse Whole Brain in being sliced 200-500um on Lycra business vibratome；
Brain tissue slice is permeated 2h in the DAPI solution of 4ug/ml by step 3；
Step 4 rinses the PI brain tissue permeated 5 times, each 10min in 0.01MPBS；
Step 5 prepares NAGA solution, and wherein initiator selects VA-044, and 4 DEG C of refrigerators save；
Step 6 will permeate 1-60min in Mice brain tissues NAGA solution；
The brain tissue permeated is put and fresh NAGA solution is added dropwise in test tube polymerize in baking oven by step 7；
Step 8, by the brain piece after polymerization in being imaged on Laser Scanning Confocal Microscope.
Can be seen that hydrogel embeds by result shown in fig. 6 can be reserved for multicolor fluorescence signal, be suitable for multicolor fluorescence
Imaging samples.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (9)
1. a kind of hydrogel embedding method for protecting mechanics of biological tissue and fluorescence characterized by comprising
Tissue is pre-processed；
NAGA monomer and initiator, solvent are mixed to get NAGA solution；
Described pretreated group is woven in the NAGA solution and carries out infiltration processing；
Group after infiltration is woven in NAGA solution and is polymerize；
It will be imaged in biological tissue's reimaging system after polymerization.
2. the method according to claim 1, wherein described pre-process tissue specifically:
Biological tissue is provided；
It is fixed after the biological tissue is carried out PFA, PBS rinsing.
3. the method according to claim 1, wherein the solvent be selected from salt, acid or alkali aqueous solution or
Pure water.
4. the method according to claim 1, wherein the mass percent of the NAGA solution is 5%-50%.
5. the method according to claim 1, wherein the initiator is selected from ammonium persulfate, potassium peroxydisulfate, azo
Diisopropyl fourth miaow hydrochloride (VA-05) or azo dicyclohexyl formonitrile HCN hydrochloride (VA-044).
6. according to the method described in claim 5, it is characterized in that, the mass percent that the initiator accounts for system is
0.01‰-5‰。
7. polymerizeing specifically the method according to claim 1, wherein the group after infiltration is woven in NAGA solution
Are as follows:
Tissue after infiltration is immersed in the NAGA solution, thermal polymerization in an oven.
8. the method according to the description of claim 7 is characterized in that the temperature of the thermal polymerization is at 25-50 DEG C, thermal polymerization
Between be 3-24h.
9. the method according to claim 1, wherein the tissue after polymerization is imaged on an imaging device specific
Are as follows:
Tissue after polymerization is placed in wide field fluorescence microscope, is imaged on Laser Scanning Confocal Microscope or Two Photon Fluorescence.
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