CN109856093A - A kind of fluorescin transgenic mouse tissue fluorescence imaging method of improvement - Google Patents
A kind of fluorescin transgenic mouse tissue fluorescence imaging method of improvement Download PDFInfo
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- CN109856093A CN109856093A CN201811563494.9A CN201811563494A CN109856093A CN 109856093 A CN109856093 A CN 109856093A CN 201811563494 A CN201811563494 A CN 201811563494A CN 109856093 A CN109856093 A CN 109856093A
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Abstract
The invention discloses a kind of fluorescin transgenic mouse tissue fluorescence imaging methods of improvement.After fixation of short duration at low temperature, by fluorescin transgenic mouse tissue after high concentration Triton is rinsed improve permeability in short-term, core is contaminated through the stronger fluorescent dye Hoescht of penetration power again, finally by the stereo-picture of scanning cofocal microscope building fluorescent protein expression.The method of the of short duration fixing organization of low temperature had both saved the activity of fluorescin to the greatest extent in the present invention, and in turn avoiding group, to be woven in subsequent experimental implementation mesometamorphism broken；High concentration Triton is rinsed in short-term increases tissue permeability, greatly improves Hoescht dye core efficiency, makes it possible that object construction positions in tissue when co-focusing imaging.By 3D common focused image constructed by the technology, signal is continuous whole, picture quality is high.It is a kind of simple and easy to do, low-cost method.
Description
Technical field
The invention belongs to cells and molecular biology and Imaging-PAM field, are related to fluorescent transgenic protein molecular and exist
Active holding and imaging method in mouse tissue.
Background technique
In traditional fluorescin transgenic mouse tissue fluorescence imaging method, with GFP transgenic mice tooth bud fluorescence at
As usually having 3 kinds of methods for technology:
(1) Stereo microscope Imaging-PAM: for the fluorescence activity for maintaining GFP, the tooth bud that will usually be obtained through dissection
Tissue block is placed in vitro tissue culture systems, and then the GFP in body formula fluorescence microscopy under the microscope entire tooth bud tissue is glimmering
Optical signal.This method is although simple and easy, but only it is observed that in tooth bud general structure fluorescence signal coarse localization, can not
Cell and observation of subcellular localization are done, and used tissue culture system needs the troublesome operations such as aseptic process.
(2) histotomy and immunofluorescence technique:, generally need to be by tooth for the cell and subcellular localization for observing fluorescin
Embryo tissue carries out paraffin section or frozen section, then carries out immunofluorescence to slice using the antibody for GFP on slice
Dyeing, then fluorescent image is obtained by Fluorescent intravital microscopy.This method acquired signals be heavily dependent on be immunized it is glimmering
The efficiency of light reaction, and the efficiency is affected by various factors, such as antibody titer and reaction system stability；Pass through the party
The obtained photo of method is discontinuous 2D flat image, can not be used to construct 3D stereopsis；And in slicing processes inevitably
Portion of tissue can be lost, dropout is caused.
(3) transgenic mice hybridization and conjugate focus imaging technique: in addition to above two common method, in order to obtain gene
The 3D rendering of cell and subcellular localization in the tissue also has researcher to attempt GFP transgenic mice tooth bud tissue culture
It is placed directly under Laser Scanning Confocal Microscope and shoots image, but nucleus or endoglin expression due to lacking display tissue basic structure
Fluorescin auxiliary positioning, under the narrow Laser Scanning Confocal Microscope visual field, so that GFP fluorescin location difficulty, pole under mirror
The difficulty taken pictures is increased greatly, and seriously affects the picture quality of photo.In order to make up this defect, also there is researcher by GFP
Transgenic mice hybridizes with the transgenic mice of expression core or film positioning fluorescin (such as RFP, YFP), to obtain double fluorescence
Then this is to be directly used in confocal microscopic image after the tooth bud anatomic tissue of mouse, obtains GFP albumen by protein expression mouse system
3D image.Although this method makes to position the reduction of GFP target protein difficulty under mirror, but a variety of transgenosis involved in this method
Mouse system maintain and mate and the high-cost experimentations such as the screening and identification of the double fluorescent protein expression mouse systems of filial generation, hence it is evident that
Hinder the popularization and application of this method.
Therefore, it is necessary to be improved to traditional method, establish it is a kind of be not only conducive to keep fluorescin activity, but also convenient for into
The positioning of row microscope undertissue, the technology for keeping subsequent 3D confocal microscopic image smooth.
Summary of the invention
In view of the various defects of above-mentioned tradition and conventional method, the present invention passes through the tissue of fluorescin transgenic mice
The of short duration fixation of low temperature is not only played the role of saving institutional framework, but also dramatically avoids the fluorescent quenching of fluorescin；Again
It is rinsed in short-term by high concentration Triton and increases tissue permeability, to improve Hoescht fluorescent dye dye core efficiency, so that tissue
Structure is high-visible, readily discernible under mirror, finally makes subsequent confocal microscopic image experiment smooth.By this
3D common focused image constructed by technology, signal is continuous whole, picture quality is high.
Technical scheme is as follows to achieve the above object:
It successively include such as in a first aspect, providing a kind of fluorescin transgenic mouse tissue fluorescence imaging method of improvement
Lower step:
(1) dissection obtains the fluorescin transgenic mouse tissue block of target gene promoter driving expression；
(2) tissue block is through 4% paraformaldehyde, 4 degree of of short duration fixations；
(3) tissue block fixed rinses in short-term through high concentration Triton；
(4) tissue block is dipped in 4 degree of dye cores in Hoescht 33342 and stays overnight；
(5) Laser Scanning Confocal Microscope Z-Stack is scanned, and constructs destination gene expression stereo-picture in tissue.
Preferably, the fluorescin transgenic mouse tissue fluorescence imaging method of above-mentioned improvement, the fluorescin are
GFP、RFP、YFP。
Further, the fluorescin transgenic mouse tissue fluorescence imaging method of above-mentioned improvement, the fluorescin
It in turn includes the following steps when the tissue is tooth bud tissue for GFP:
(1) dissection obtain target gene promoter driving GFP transgenosis new life to be born after 7 days mouse (P0-P7) cut
Tooth end flap block (tooth bud tissue block) is soaked in 4% paraformaldehyde after rinsing at room temperature in 1 × PBS, is placed in 4 degree and is delayed
Slow oscillation fixes 5 minutes, after the completion of fixed, rinses at room temperature in 1 × PBS；
(2) tissue block fixed is rinsed in short-term in 1 × PBS-0.5%Triton, to improve tissue permeability, institute
It states 1 × PBS-0.5%Triton and refers to that Triton is dissolved in 1xPBS, final concentration of the 0.5% of Triton
(3) tooth bud tissue block is soaked in final concentration of 5 μ g/mL fluorescent dye Hoescht 33342 again, is placed in 4 degree
Slow oscillation, dye core are stayed overnight, after the completion of contaminating core, are rinsed at room temperature in 1 × PBS；
(4) tissue block of above-mentioned processing is placed in the glass slide groove of the groove containing self-control, one is instilled in groove
Then drop Vectashield infiltration tissue block covers coverslip as the refractive power medium of co-focusing imaging on groove；
(5) Z-Stack scanning is carried out to tissue block using Laser Scanning Confocal Microscope, and constructs tooth bud tissue using acquired image
The stereopsis of destination gene expression in block.
Beneficial effects of the present invention:
1. the present invention not only saves the activity of fluorescin, but also avoid by the of short duration fixing organization of low temperature to the greatest extent
It is broken that group is woven in subsequent experimental implementation mesometamorphism；
2. directly contaminate core after the pioneering of short duration fixation of low temperature, becoming object construction positioning can
Energy.
3. the core dyestuff Hoescht for selecting penetration into tissue strong, and rinsed in short-term before contaminating core using high concentration Triton,
To increase tissue to the permeability of dyestuff, dye core efficiency is greatly improved.
4. the step of eliminating the tissue cultures in Stereo microscope Imaging-PAM, and the present invention can obtain biography
The cell and subcellular localization 3D rendering of the unobtainable GFP fluorescin of system method 1；
5. the step of eliminating histotomy and Immunofluorescence Reactions in histotomy and immunofluorescence technique, when saving
Between, save that cost, imaging effect are more preferable, and compensate for histotomy dropout and be only capable of obtaining the defect of 2D flat image；
6. eliminating a variety of fluorescin transgenic mouse lines dimensions in transgenic mice hybridization and conjugate focus imaging technique
The complexity of system and hybridization and filial generation screening and identification, expensive experimental procedure.
Detailed description of the invention
In Fig. 1 .Lgr5-GFP transgenosis P2 mouse incisor end flap in Stem Cell Niche GFP fluoroscopic image.(scale bar
=50 μm)
A, incisor stem cell can only finding Lgr5-GFP expression and distribution mode under body formula fluorescence microscope: be recognized from the image
The approximate range of nest, the fluorescence signal of Lgr5-GFP is faintly shown in the outside of incisor Stem Cell Niche, but can not judge fluorescence
The cell in signal source and the subcellular localization of signal.
B, the incisor shot under Laser Scanning Confocal Microscope using P2 mouse incisor tissue block sample prepared by the method for the present invention
Stem Cell Niche Z-Stack image: Hoechst33342 dye core (blue) makes the clear in structure as it can be seen that visible simultaneously of Stem Cell Niche
Apparent core and slurry expression pattern is presented in the outside of Stem Cell Niche in the signal distributions of Lgr5-GFP (green).
C, Z-Stack is carried out under Laser Scanning Confocal Microscope using P2 mouse incisor tissue block sample prepared by the method for the present invention
After scanning, the incisor Stem Cell Niche Lgr5-GFP 3D of building expresses image.
In Fig. 2 .Sox2-GFP transgenosis P2 mouse incisor end flap in Stem Cell Niche GFP fluoroscopic image.
(scale bar=50 μm)
A, finding Sox2-GFP expression and distribution mode under body formula fluorescence microscope: from the image as it can be seen that Sox2-GFP's is glimmering
Optical signal is distributed in entire incisor Stem Cell Niche, keeps the structure of Stem Cell Niche readily discernible, but still can not judge fluorescence signal
The cell in source and the subcellular localization of signal.
B, the incisor shot under Laser Scanning Confocal Microscope using P2 mouse incisor tissue block sample prepared by the method for the present invention
Stem Cell Niche Z-Stack image: Hoechst33343 dye core (blue) makes the clear in structure as it can be seen that visible simultaneously of Stem Cell Niche
Core and slurry expression pattern is presented in entire Stem Cell Niche in the signal distributions of Sox2-GFP (green).
C, Z-Stack is carried out under Laser Scanning Confocal Microscope using P2 mouse incisor tissue block sample prepared by the method for the present invention
After scanning, the incisor Stem Cell Niche Sox2-GFP 3D of building expresses image.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.[embodiment 1] determines
Fixed temperature, set time, Triton concentration, dye core dyestuff and dye in GFP transgenic mice tooth bud histofluorescence imaging method
The process of core time is as follows:
(1) fixed temperature and set time: by P2GFP transgenic mice incisor end flap's block in 4% paraformaldehyde
At room temperature or 4 degree are fixed 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes respectively.It redyes and is imaged subsequent
In the process, 4 degree of tissues for fixing 5 minutes, tissue integrity and GFP fluorescence signal intensity are best；Room temperature fixation makes fluorescence quench
It goes out more；Set time is shorter than 5 minutes, cannot have the function that keep tissue integrity, organize cracky；Set time is longer than
Fluorescent quenching in 5 minutes is more.Therefore be suitable for fixed temperature and set time be 4 degree 5 minutes.
(2) tissue block after fixation Triton concentration: is placed in 1 × PBS-0.1%Triton, 1 × PBS-0.5%
It is rinsed in Triton, 1 × PBS-1.0%Triton 5 minutes × 2 times, 0.5%Triton is improving tissue permeability as the result is shown
While, do not destroy tissue integrity；And 0.1% conventional Triton concentration cannot effectively improve the permeability of tissue block, make
Subsequent Hoechst33342 dyeing efficiency substantially reduces；And although higher concentration 1.0%Triton improves tissue permeability, but
Tissue integrity is obviously destroyed simultaneously, tissue damage is serious in subsequent experimental.Therefore suitable Triton concentration is 0.5%.
(3) dye core dyestuff and dye core time: incisor end flap block is respectively placed in two kinds of fluorescent dye DAPI after fixation
In Hoechst33342,4 degree of lower progress nuclear stainings are stayed overnight for dyeing duration 2 hours, 4 hours, 8 hours, 16 hours.As a result it shows
Show that the penetrability of Hoechst33342 is preferable, when dyeing continued overnight, tissue block outer layer and centrocyte present uniformly clear
Clear core colored fluorescent signal, and when Hoescht33342 dyeing time is shorter than stayed overnight, the dyeing of tissue block centrocyte is endless
Entirely；And DAPI is dyed, and under the conditions of a variety of durations tested, is all only capable of making the nucleus on tissue block surface layer to colour, tissue block
Unstressed configuration signal in centrocyte.It therefore is suitable for contaminating core dyestuff and contaminating the core time for Hoechst33342 stained over night.
The confocal fluorescent of [embodiment 2] Lgr5-GFP transgenic mice incisor tooth bud tissue is imaged
1, material and method
1.1 material
Animal: (P2) on the 2nd one tires of Lgr5-GFP transgenic mice after birth (6-8 is only)；
Reagent: 1 × PBS, 1 × PBS-0.5%Triton, 10mg/mL Hoechst 33342, Vestsheild；
Consumptive material and instrument: glass slide, coverslip, Laser Scanning Confocal Microscope.
1.2 method
(1) dissection obtains Lgr5-GFP transgenosis P2 mouse incisor end flap block, the slowly vibration at room temperature in 1 × PBS
After swinging rinsing 5 minutes × 2 times, it is soaked in 4% paraformaldehyde, is placed in 4 degree of slow oscillations, 5 minutes, after having fixed are fixed, in 1
Slow oscillation rinses 5 minutes × 2 times at room temperature in × PBS；
(2) tissue block after fixation is soaked in 1 × PBS-0.5%Triton, slow oscillation rinses 5 minutes × 2 times；
(3) the tooth bud tissue block through handling above is soaked in final concentration of 5 μ g/mL fluorescent dye Hoescht33342
In, it is placed in 4 degree and is protected from light slow oscillation, dye core is stayed overnight, after the completion of contaminating core, rinsed at room temperature in 1 × PBS 5 minutes × 2 times；
(4) tissue block is placed in the glass slide groove of the groove containing self-control, adjustment tissue Block direction to most right position is set, recessed
A drop Vectashield is instilled in slot and infiltrates tissue block, and as the refractive power medium of co-focusing imaging, lid is then covered on groove
Slide；
(5) Z-Stack scanning is carried out to tissue block using Laser Scanning Confocal Microscope, and constructs tooth bud using acquired image
The stereopsis of Lgr5-GFP expression.
(4) result
The nucleus (Hoechst33332 coloring) of indigo plant dye is high-visible, keeps Stem Cell Niche structure readily discernible；Green
Nuclear expression is presented in the outside of Stem Cell Niche, and in part in Lgr5-GFP signal Assembled distribution into the cell, and intracellular in part
In slurry expression pattern.
Therefore, this method can high-efficient simple the confocal microscopic image for realizing Lgr-GFP fluorescin in tissue, and institute
The reagent used is inexpensive common biochemical reagents, substantially reduces imaging cost.
The confocal fluorescent of [embodiment 3] Sox2-GFP transgenic mice incisor tooth bud tissue is imaged
1, material and method
1.1 material
Animal: (P2) on the 2nd one tires of Sox2-GFP transgenic mice after birth (6-8 is only)；
The other the same as in Example 1.
1.2 method
(1) dissection obtains Sox2-GFP transgenosis P2 mouse incisor end flap block.
(2) the other the same as in Example 1.
(4) result
The nucleus (Hoechst33342 coloring) of indigo plant dye is high-visible, keeps Stem Cell Niche structure readily discernible；Green
Sox2-GFP signal is uniformly distributed in entire Stem Cell Niche most cells, and then in apparent core and slurry expression pattern.
Claims (3)
1. a kind of fluorescin transgenic mouse tissue fluorescence imaging method of improvement, which is characterized in that successively include following step
It is rapid:
(1) dissection obtains the fluorescin transgenic mouse tissue block of target gene promoter driving expression；
(2) tissue block is through 4% paraformaldehyde, 4 degree of of short duration fixations；
(3) tissue block fixed rinses in short-term through high concentration Triton；
(4) tissue block is dipped in 4 degree of dye cores in Hoescht 33342 and stays overnight；
(5) Laser Scanning Confocal Microscope Z-Stack is scanned, and constructs destination gene expression stereo-picture in tissue.
2. the fluorescin transgenic mouse tissue fluorescence imaging method of improvement according to claim 1, which is characterized in that
The fluorescin is any one in GFP, RFP, YFP.
3. the fluorescin transgenic mouse tissue fluorescence imaging method of improvement according to claim 2, which is characterized in that
The fluorescin is that GFP in turn includes the following steps when the tissue is tooth bud tissue:
(1) mouse incisor end flap on the 7th after the GFP transgenosis new life that dissection obtains the driving of target gene promoter is extremely born
Block, i.e. tooth bud tissue block are soaked in 4% paraformaldehyde after rinsing at room temperature in 1 × PBS, are placed in 4 degree of slow oscillations, Gu
It is 5 minutes fixed, after the completion of fixed, rinsed at room temperature in 1 × PBS；
(2) tissue block fixed is rinsed in short-term in 1 × PBS-0.5%Triton；
(3) tooth bud tissue block is soaked in final concentration of 5 μ g/mL fluorescent dye Hoescht 33342 again, be placed in 4 degree it is slow
Oscillation contaminates core 16 hours, after the completion of contaminating core, rinses at room temperature in 1 × PBS；
(4) tissue block of above-mentioned processing is placed in the glass slide groove of the groove containing self-control, a drop is instilled in groove
Vectashield infiltrates tissue block as the refractive power medium of co-focusing imaging and then covers coverslip on groove；
(5) Z-Stack scanning is carried out to tissue block using Laser Scanning Confocal Microscope, and using in acquired image building tooth bud tissue block
The stereopsis of destination gene expression.
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| CN111311653A (en) * | 2020-02-10 | 2020-06-19 | 杭州电子科技大学 | Method for registering dental plaque fluorescence image and tooth three-dimensional model |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8159676B2 (en) * | 2008-01-21 | 2012-04-17 | University Of North Texas, Health Science Center At Fort Worth | Ratiometric surface plasmon coupled emission detector |
| CN105738182A (en) * | 2016-02-25 | 2016-07-06 | 河南中医学院 | Fluorescent staining method for observing plant microstructure |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8159676B2 (en) * | 2008-01-21 | 2012-04-17 | University Of North Texas, Health Science Center At Fort Worth | Ratiometric surface plasmon coupled emission detector |
| CN105738182A (en) * | 2016-02-25 | 2016-07-06 | 河南中医学院 | Fluorescent staining method for observing plant microstructure |
Non-Patent Citations (1)
| Title |
|---|
| HARIHARAN P. EASWARAN等: "Cell Cycle Markers for Live Cell Analyses", 《LANDES BIOSCIENCE》 * |
Cited By (2)
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|---|---|---|---|---|
| CN111311653A (en) * | 2020-02-10 | 2020-06-19 | 杭州电子科技大学 | Method for registering dental plaque fluorescence image and tooth three-dimensional model |
| CN111311653B (en) * | 2020-02-10 | 2023-04-21 | 杭州电子科技大学 | Method for registering dental plaque fluorescent image and tooth three-dimensional model |
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