CN109406768A - A method of the distribution of observation microtissue inner cell solid and statistics cell number - Google Patents
A method of the distribution of observation microtissue inner cell solid and statistics cell number Download PDFInfo
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- CN109406768A CN109406768A CN201811311740.1A CN201811311740A CN109406768A CN 109406768 A CN109406768 A CN 109406768A CN 201811311740 A CN201811311740 A CN 201811311740A CN 109406768 A CN109406768 A CN 109406768A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Abstract
The present invention relates to observation microtissue inner cell solid distribution a kind of in the microscopic observation technical field of biological tissue and the method for statistics cell number, the organization material to be seen that will acquire successively carries out chemical fixation, dehydration, immunofluorescence dyeing and transparency process after carrying out surface clean；Organization material is moved into Laser Scanning Confocal Microscope special culture dish together with transparent liquid again, successively scan with laser confocal microscope and take pictures, obtain the cell distribution image of each layer of global tissue, carry out the observation of cell solid distribution, each confluent monolayer cells distributed image data are handled by Imaris software again, the threedimensional model of reconstituted cell tissue, it observes in three-dimensional stereo model and is distributed by the solid of immunofluorescence label cell, it recycles Imaris Bitplane Spot to test and analyze, calculates the exact amount of immunofluorescence label cell.Method of the invention maintains the integrality of tissue to be seen, and the clarity of observation cell distribution and accuracy are all high, and the number statistical result of cell is more acurrate.
Description
Technical field
The present invention relates to the microscopic observation technical field of biological tissue, in particular to a kind of observation microtissue inner cell is vertical
The method of body distribution and statistics cell number.
Background technique
In life science and Clinicopathological analysis, it is thin to observe the micro Distribution of cell and statistics in biological tissue
Born of the same parents' number has very important significance to research cell differentiation, tissue development, disease generation etc..Study the normal of this process
Rule method is after carrying out serial section to tissue, using immunohistochemical staining, then to pass through the picture number between different slices
The distribution of cell in complete tissue is simulated and deduced according to splicing and superposition, and then calculates the number of specific cells.But this
The limitation of one conventional method is that serial section can not show really tissue overall picture, cell distribution and specific cells completely
Quantity.Histotomy is only able to display the cell distribution and quantity of some level, moreover in actual fabrication slice, is difficult to avoid
To disorganization and loss, the accuracy of single slice cell distribution and quantity not can guarantee, it could be also difficult to guarantee difference
The homogeneity of slice thickness, this results in superimposed result more inaccurate, is even rebuild and is calculated with mathematical model,
Its final result is also only predicted and estimation, not true image and data, this results in same biological tissue
The variable coefficient of sample, final result is very big, and the result credibility of especially cell quantity is not high.
How while keeping tissue integrity, the distribution and quantity in observation specific cells portion within the organization are biologies
Learn the emphasis and hot spot of research and medical research.Although the present common computed tomography of clinical medicine (CT), just
Electron emission computed tomography (PETCT), the technologies such as Magnetic resonance imaging (MRI) are developed rapidly, but at present
Until, cellular level or subcellsular level is still not achieved in resolution ratio.
Summary of the invention
It is an object of the present invention to provide the distribution of microtissue inner cell solid is observed while a kind of holding tissue integrity
And the method for statistics cell number, one kind is provided in cellular level or subcellular for life science and Clinicopathological analysis
Method level observation tissue and count cell quantity.
Above-mentioned purpose to realize the present invention, present invention firstly provides a kind of observation microtissue inner cell solid distributions
Method, comprising the following steps: obtain organization material to be seen and carry out surface clean；To acquired organization material
Learn fixed and dehydration；Immunofluorescence dyeing is carried out to the organization material after dehydration；To the tissue of immunofluorescence dyeing
Material carries out transparency process；The organization material of transparency process is moved into the dedicated culture of Laser Scanning Confocal Microscope together with transparent liquid
Ware successively scan with laser confocal microscope and be taken pictures, obtains the cell distribution image of each layer of global tissue, carry out cell
The observation of solid distribution.
Method of the invention, by carrying out chemical fixation, dehydration, immunofluorescence dyeing and thoroughly to whole biological tissue
Brightization processing, keeps the integrality of the whole biological tissue obtained, carries out identification and fluorescence mark to the specific cell in tissue
Then note carries out successively scanning by laser confocal microscope and takes pictures, obtain the cell of each layer of global tissue in order to observe
Distributed image carries out the observation of cell solid distribution.In method of the invention to the disposed of in its entirety of biological tissue and observation process,
The integrality for keeping tissue entirety does not need hierarchy slicing observation, realizes the high-resolution essence of cellular level or subcellsular level
Really observation.
It is described layer-by-layer to scan between the thickness taken pictures as a preference of the present invention, the thickness of the organization material is less than 500 μm
Away from being 1~5 μm.
The chemical fixing step of organization material of the invention includes: that the chemical fixing step of organization material includes: 2~5
Under conditions of DEG C by the organization material after flushing be immersed in mass concentration be fix 2 in 3.5~4.5% paraformaldehyde aqueous solutions~
2.5 hour；Later, remove paraformaldehyde aqueous solution, under conditions of 2~5 DEG C, organization material is used respectively phosphate buffer into
Row oscillation elution 3~4 hours, the phosphate buffer was primary every replacement in 1 hour.By the processing of this step, reduce in tissue
The water solubility of many kinds of substance such as protein, peptide matters guarantees global tissue in order to which organization material is carried out hardening fixation
Integrality.
For the thorough dehydration for being easy to implement organization material inner cell, dehydration process steps of the invention include: to remove dehydration
Organization material mass concentration is 9~11% Hes under conditions of 2~5 DEG C by phosphate buffer in treated organization material
19~21% aqueous sucrose solution oscillation elution 3~4 hours；Later, continue organization material quality under conditions of 2~5 DEG C
The aqueous sucrose solution oscillation that concentration is 29~31% elution 12-15 hours.
Further, immunofluorescence dyeing step of the invention includes: the sucrose solution removed after dehydration, by tissue
Material successively carries out that permeable membrane processing and Seal treatment, then to be successively incubated for 48~72 with the secondary antibody of primary antibody and fluorescent marker respectively small
When, fluorescent staining is carried out finally by fluorescent dye.
Further, permeable membrane treatment process of the invention are as follows: under conditions of 20~25 DEG C, by organization material in permeable membrane liquid
Middle oscillation incubation 12~15 hours, the permeable membrane liquid are that the Triton X-100 that mass concentration is 0.4~0.6% is water-soluble
Liquid.The permeable membrane treatment process of this step can be improved the permeability of cell membrane of organization material by the control of permeable membrane time, and make
Cell reaches same permeability of cell membrane inside complete tissue；So as to antibody (including primary antibody and secondary antibody) in subsequent processes
Cell inside complete tissue can be can smoothly enter into.
Further, the Seal treatment process in the method for the present invention are as follows: remove the permeable membrane in the organization material of permeable membrane processing
Liquid, under conditions of 4~5 DEG C, by organization material oscillation incubation 12~15 hours, closing in confining liquid of permeable membrane processing
Liquid ingredient includes following quality component aqueous solution: 0.4~0.6% Triton X-100,0.10~0.20% sweet ammonia
Acid, 9~11% fetal calf serum, 3~4% bovine serum albumin(BSA).It is handled by this step, can reduce in subsequent processes and resist
The non-specific binding of body eliminates the generation of false positive signal.
For convenient for identifying certain a kind of specific cell, primary antibody of the invention and secondary antibody incubation process include:
The organization material of Seal treatment is moved into primary antibody solution under the conditions of 4~5 DEG C by the confining liquid for S1) removing Seal treatment
In, it is incubated for 48~72 hours with the revolving speed shaking table of 50~60rpm；Keep certain in primary antibody and biological tissue a kind of thin by primary antibody incubation
The specifically expressed albumen of born of the same parents combines, to achieve the purpose that identify certain a kind of cell in tissue；
S2 primary antibody solution) is removed, under the conditions of 2~5 DEG C, the Triton X-100 for being 0.1% with mass concentration is water-soluble
Liquid is cleaned, and is washed 3~4 hours with the revolving speed shaking table of 50~60rpm, and every the aqueous solution of replacement in 1 hour；Pass through this
Step processing can wash the primary antibody not specifically bound；
S3) the aqueous cleaning solution walked in removal moves into organization material in two corresponding anti-solution, under the conditions of being protected from light for 2~5 DEG C, with 50
The revolving speed shaking table of~60 rpm is incubated for 48~72 hours, if S1) in primary antibody be coupled with fluorophor, this step omission；This step
Purpose be with the secondary antibody of fluorescent marker to S1) in primary antibody carry out Immune discrimination, in order to aobvious by fluorescence in subsequent step
Micro mirror can tell specific cell；
S4 two corresponding anti-solution) is removed, under the conditions of 42~5 DEG C, organization material is moved into the polyethylene glycol octyl of mass concentration 0.1%
Phenyl ether aqueous solution is cleaned, and is washed 3~4 hours with the revolving speed shaking table of 50~60rpm, and every the water of replacement in 1 hour
Solution, if S3) it omits, this step is omitted；
S5) the cleaning solution walked in removal moves into organization material in DAPI dilution under the conditions of being protected from light for 2~5 DEG C, with 50~
The revolving speed shaking table of 60 rpm is incubated for 10~12 hours, to identify that all cells in tagged tissue, the DAPI dilution are 4', 6-
Diamidino -2-phenylindone dilute aqueous solution；By this step process, DAPI molecule can be made and organize the DNA of interior all cells
In conjunction in order to observe point of the specific cell of cell distribution and preceding step antibody identification label all in tissue within the organization
Cloth.
Further, the transparency process step includes: to move into the organization material of immunofluorescence dyeing in transparent liquid,
It under the conditions of being protected from light for 20~25 DEG C, is incubated for 48~72 hours with the revolving speed shaking table of 50~60 rpm, the transparent liquid is following matter
Measure the aqueous solution of component: 22~26% urea, 8~12% glycerol, 0.9~0.12% Triton X-100,48
~52% sucrose.By the processing of this step, laser penetration tissue surface can be made, into organization internal, so that micro-
Mirror can identify the fluorescence signal inside complete tissue.
Method of the invention is keeping biological tissue to be seen by biological tissue to be seen by above-mentioned processing
While integrality, fluorescent marker is carried out to certain a kind of cell in tissue by specific antibody, after transparency process,
Using the fluorescence signal of laser confocal microscope identification complete tissue whole, the thin of specific marker really and is three-dimensionally shown
Spatial distribution of the born of the same parents in complete tissue.The present invention solves the fluorescent marker of cell and microexamination inside complete tissue, will
The cellular level and three-dimensional level that the observation of complete tissue improves；Theoretically, being had by means of high-resolution confocal microscope can
Subcellsular level or molecular level can be reached.Especially in terms of transparency process, the present invention exempts from before transparency process
Epidemic disease fluorescent staining avoids transparency process to the damage of antigen and the loss or reduction of fluorescence signal resulting from；Meanwhile
The transparent liquid of optimization makes laser be easier to penetrate complete tissue, so that fluorescence signal is clear and background signal is weak.
It is a further object to provide a kind of methods for counting microtissue inner cell number, first will be by above-mentioned
The method for observing the distribution of microtissue inner cell solid obtains the cell distribution image of each layer of microtissue, passes through Imaris software
Each confluent monolayer cells distributed image data are handled, histiocytic threedimensional model is rebuild, are observed in three-dimensional stereo model by immunofluorescence
Mark the three-dimensional distribution of cell that it is thin to calculate immunofluorescence label by testing and analyzing using Imaris Bitplane Spot
The exact amount of born of the same parents.Cell number statistical method through the invention passes through according to the cell distribution maps of each layer of microtissue
Imaris three-dimensional software reproduce microtissue cell distribution high definition wash rice degree, the threedimensional model of high validity, with accurately count to
The cell number of the whole biological tissue of observation.
Detailed description of the invention
Fig. 1 be 1 day-old Mice obtained using the method for observation microtissue inner cell solid distribution of the invention from
The full tissue scanning layer figure of the laser co-focusing of body gonadal tissue.
Fig. 2 is the local screenshot of the partial scan layer figure in Fig. 1, wherein left side vertical setting of types is with TRA98 to complete in vitro
Reproduction cell specific marker in gonadal tissue is the scanning figure of green (white is had been converted into figure), and intermediate vertical setting of types is to use
DAPI is identified as the tracing of beating of blue (grey is had been converted into figure) to all cells in complete in vitro gonadal tissue, and right side is perpendicular
Row is the figure of all cell distributions of the global tissue after left bank and intermediate row conjunction figure.
Fig. 3 is the three-dimensional rotation figure that the in vitro gonadal tissue of synthesis is reproduced by Imaris software.
Fig. 4 is the three dimensional pattern by Imaris software according to the special sexual reproductive cell building of fluorescent marker in Fig. 1-3
Figure.
Specific embodiment
The present embodiment is by taking the in vitro sexual gland for a day-old Mice of being born as an example, in conjunction with the attached drawing substep side that the present invention will be described in detail
Method.
One, by the in vitro gonadal tissue of a day-old Mice, through PBS(phosphate buffer) or brine to wash away group
Knit the inessential tissue of material surface, the thickness of the in vitro gonadal tissue of the day-old Mice obtained in this example less than 500 μm,
Through being measured under microscope, with a thickness of 228 μm.
Two, chemical fixation and dehydration are carried out in vitro tissue
1. by upper step, treated that in vitro tissue is transferred in 1.5 ml centrifuge tubes, and the mass concentration that 1 ml Fresh is added is
4% paraformaldehyde aqueous solution, it is fixed under conditions of 4 DEG C to be incubated for 2 hours；
2. carefully removing the paraformaldehyde aqueous solution after fixing process, use phosphate buffer with 500 rpm respectively in vitro tissue
Revolving speed shaking table wash 3 hours, phosphate buffer is primary every replacement in 1 hour, after aforementioned washing, removes residual in vitro tissue
The paraformaldehyde solution stayed；
3. carefully removing phosphate buffer in vitro tissue, it is 10% that 1 ml mass concentration is added into the centrifuge tube of in vitro tissue
Aqueous sucrose solution is washed 3 hours under the conditions of 4 DEG C with the revolving speed shaking table of 50 rpm；
4. carefully removing above-mentioned mass concentration is 10% aqueous sucrose solution, it is dense that 1 ml mass is added into the centrifuge tube of in vitro tissue
Degree is 20% aqueous sucrose solution, is washed 3 hours under the conditions of 4 DEG C with the revolving speed shaking table of 50 rpm；
5. carefully removing above-mentioned 20% aqueous sucrose solution of mass concentration, it is 30% that mass concentration is added into the centrifuge tube of in vitro tissue
Aqueous sucrose solution is washed 12 hours under the conditions of 4 DEG C with the revolving speed shaking table of 50 rpm.
After the washing of above-mentioned three steps aqueous sucrose solution, the thorough dehydration of cell inside complete tissue may be implemented.
Three, immunofluorescence dyeing is carried out in vitro tissue
1. permeable membrane is handled, the in vitro tissue after above-mentioned fixation and dehydration is moved into the centrifuge tube that another capacity is 1.5 ml
In, the aqueous solution (Triton X-100 aqueous solution) of 0.5 Triton X-100 of mass concentration is added, in 25 DEG C of items
With the revolving speed shaking table oscillation incubation of 50 rpm 12 hours under part；It is handled by this step permeable membrane, histiocytic cell can be improved
Membrane permeability, and cell inside complete tissue is made to reach same permeability of cell membrane；So as to anti-in subsequent processes
Body (including primary antibody and secondary antibody) can smoothly enter into cell inside complete tissue.
2. Seal treatment carefully removes above-mentioned permeable membrane liquid, 1ml confining liquid is added, is turned under the conditions of 4 DEG C with 50 rpm
Fast shaking table oscillation incubation 12 hours, confining liquid used be following quality component aqueous solution: 0.5% Triton X-100(namely
Triton X-100), 0.15% glycine, 10% fetal calf serum, 3% BSA(bovine serum albumin(BSA))；Pass through
This step Seal treatment can reduce the non-specific binding of antibody in subsequent processes, eliminate the generation of false positive signal.
3. primary antibody is incubated for, the confining liquid after carefully removing Seal treatment, it is 2 μ that 0.5ml concentration is added into vitro tissue
The TRA98 primary antibody solution of g/ml is incubated for 48 hours under the conditions of 4 DEG C with the revolving speed shaking table of 50 rpm, for in vitro tissue
Reproduction cell specific antigen combines, and the primary antibody TRA98 of the present embodiment is Anti-Germ cell- of the purchase from abcam company
Specific antigen antibody；
4. carefully remove primary antibody solution, Triton X-100 aqueous solution (namely the polyethylene glycol octyl for being 0.1% with mass concentration
Phenyl ether aqueous solution) it is cleaned, it under the conditions of 4 DEG C, is washed 3 hours with the revolving speed shaking table of 50rpm, and replaced every 1 hour
Aqueous solution；
5. walking cleaning solution on removing, organization material is moved into two corresponding anti-solution, under the conditions of being protected from light for 4 DEG C, with turning for 50rpm
Fast shaking table is incubated for 48 hours, and the secondary antibody in this step is goat anti-rat IgG H&L (Alexa of the purchase from abcam company
Fluor 488), in conjunction with primary antibody TRA98, if the primary antibody in 3 is coupled with fluorophor, this step to be omitted；This step
Purpose is that the primary antibody without fluorophor is identified and marked, in order to which laser confocal microscope identification specificity is thin
Born of the same parents；
6. removing secondary antibody melt, under the conditions of 4 DEG C, the Triton X-100 that organization material is moved into mass concentration 0.1% is water-soluble
Liquid (namely Triton X-100 aqueous solution) is cleaned, and is washed 3 hours with the revolving speed shaking table of 50rpm, and small every 1
Aqueous solution of Shi Genghuan, if above-mentioned step 5 is omitted, this step is omitted；
7. organization material is moved into the DAPI that concentration is 1 μ g/ml and (divided by the cleaning solution walked on removing under the conditions of being protected from light for 4 DEG C
Minor is 4', 6- diamidino -2-phenylindone fluorescent dye) in melt, it is incubated for 12 hours with the revolving speed shaking table of 50rpm；
By this step process, all cell combinations in DAPI molecule and in vitro tissue can be made, it is all in tissue in order to observe
The distribution of the specific cell of cell distribution and preceding step identification label within the organization.
Four, transparency process is carried out in vitro tissue: the in vitro tissue of immunofluorescence dyeing is moved into another 1.5 newly
In ml centrifuge tube, the transparent liquid of 1 ml is added, is incubated for 48 hours under the conditions of being protected from light for 25 DEG C with the revolving speed shaking table of 50rpm.Wherein
Transparent liquid be following quality proportioning aqueous solution: containing 24% urea, 10% glycerol, 0.1% the poly- second of Triton X-100(
Glycol octyl phenyl ether), 50% sucrose.By the transparency process of this step, laser penetration tissue surface can be made, into group
Inside is knitted, so that laser confocal microscope can identify the fluorescence signal inside complete tissue.At existing transparence
Unlike reason, transparency process of the invention is carried out after immunofluorescence dyeing, avoids transparency process confrontation
The loss or reduction of former damage and fluorescence signal resulting from；Meanwhile the transparent liquid of optimization makes laser be easier to penetrate
Complete tissue, so that fluorescence signal is clear and background signal is weak.
The in vitro tissue of above-mentioned transparency process is moved in Laser Scanning Confocal Microscope special culture dish together with transparent liquid, the training
Support ware with a thickness of 0.1mm, carries out successively scanning with laser confocal microscope and takes pictures, when scanning by along Z axis with 2 μm of scanning
Spacing carry out successively scanning takes pictures, obtain each layer of global tissue full cell distribution image, as shown in Figure 1, the present embodiment from
Body tissue, with a thickness of 228 μm, has carried out 114 layers of scanning altogether and has taken pictures along Z axis, and the Arabic numerals number in figure indicates scanning
The number of plies, because diagram has been converted into artwork master, the white portion in Fig. 1 shows that cell is the reproduction cell of glimmering big gun mark.Fig. 2 is
The the 5th, 10,30,64,90 layer in Fig. 1 of scanned picture is intercepted respectively, and wherein the white cell in figure in each figure of left side vertical setting of types is
The laser scanning figure of the equivalent layer of fluorescence labelling is carried out to the reproduction cell in complete in vitro tissue with TRA98 antibody, it is intermediate
Vertical setting of types is the laser scanning figure that with DAPI all cells in complete in vitro tissue are carried out with the equivalent layer of fluorescent staining, and right side is perpendicular
Row is the figure that left vertical setting of types and intermediate vertical setting of types are superimposed all cell distributions of the global tissue of equivalent layer after closing figure.Pass through the series of drawing
The distribution situation of observation global tissue internal specific reproduction cell that can be comprehensive, can be seen that from the figure that left side is respectively arranged
With TRA98 be identified as green single reproduction cell it is high-visible, be clearly outlined, boundary it is clearly demarcated, gonadal tissue periphery and
Internal visible single reproduction cell, and reproduction cell is largely distributed in the periphery of gonadal tissue, and gonadal tissue is interior
Portion's (medullary substance) distribution is less.The above-mentioned processing mode to the in vitro gonadal tissue of mouse through this embodiment, comprehensive can see
It examines and understands true cell distribution inside in vitro tissue.
For the quantity convenient for reproduction cell in accurate statistics in vitro tissue, each confluent monolayer cells picture number that the above process is obtained
Handled according to by Imaris software, rebuild the threedimensional model of in vitro tissue, carry out 3 D stereo rotational view, further appreciate that by
The distribution of the reproduction cell of mark within the organization, as shown in figure 3, threedimensional model rotated clockwise respectively along Y-axis 0 °, 45 °, 90 °,
135 ° and 180 ° of tri-dimensional picture.
By testing and analyzing using Imaris Bitplane Spot, the accurate number of immunofluorescence label cell is calculated
Mesh.Cell number statistical method through the invention, it is soft by Imaris three-dimensional according to the cell distribution maps of each layer of microtissue
Part reproduces the high definition wash rice degree of microtissue cell distribution, and the threedimensional model of high validity is raw with the entirety for accurately counting to be seen
The cell number of object tissue, as shown in figure 4, passing through the three-dimensional mould for a threedimensional model wherein screenshot for reproduction cell building
Type carries out multi-directional rotation and observes statistical testing of business cycles, and the exact amount of reproduction cell is 2415.
The invention is not limited to the above-mentioned embodiments to the in vitro gonadal tissue of mouse, can be used for observation and handle it
Its microtissue material.
Claims (10)
1. a kind of method of observation microtissue inner cell solid distribution, which comprises the following steps: obtain to be seen
Organization material and carry out surface clean；Chemical fixation and dehydration are carried out to acquired organization material；To dehydration
Organization material afterwards carries out immunofluorescence dyeing；Transparency process is carried out to the organization material of immunofluorescence dyeing；By transparence
The organization material of processing moves to Laser Scanning Confocal Microscope special culture dish together with transparent liquid, is carried out successively with laser confocal microscope
Scanning is taken pictures, and the cell distribution image of each layer of global tissue is obtained, and carries out the observation of cell solid distribution.
2. the method for observation microtissue inner cell solid distribution according to claim 1, which is characterized in that the tissue
For the thickness of material less than 500 μm, the layer-by-layer thickness spacing taken pictures that scans is 1~5 μm.
3. the method for observation microtissue inner cell solid distribution according to claim 1, which is characterized in that organization material
Chemical fixing step include: under conditions of 2~5 DEG C by the organization material after flushing be immersed in mass concentration be 3.5~
2~2.5 hours are fixed in 4.5% paraformaldehyde aqueous solution；Later, paraformaldehyde aqueous solution is removed, under conditions of 2~5 DEG C,
Organization material is subjected to oscillation elution 3~4 hours with phosphate buffer respectively, the phosphate buffer replaced one every 1 hour
It is secondary.
4. the method for observation microtissue inner cell solid distribution according to claim 1, which is characterized in that the dehydration
Processing step include: remove dehydration after organization material in phosphate buffer, under conditions of 2~5 DEG C, by organization material
Aqueous sucrose solution oscillation elution 3~4 hours for being 9~11% and 19~21% with mass concentration；Later, continue the item at 2~5 DEG C
Aqueous sucrose solution oscillation elution 12-15 hours for being 29~31% by organization material mass concentration under part.
5. the method for observation microtissue inner cell solid distribution according to claim 1, which is characterized in that immunofluorescence
Staining procedure includes: the sucrose solution removed after dehydration, and organization material is successively carried out to permeable membrane and is handled with Seal treatment, so
It is successively incubated for the secondary antibody of primary antibody and fluorescent marker 48~72 hours respectively afterwards, carries out fluorescent staining finally by fluorescent dye.
6. the method for observation microtissue inner cell solid distribution according to claim 5, which is characterized in that the permeable membrane
Treatment process are as follows: under conditions of 20~25 DEG C, by organization material in permeable membrane liquid oscillation incubation 12~15 hours, the permeable membrane
Liquid is the Triton X-100 aqueous solution that mass concentration is 0.4~0.6%.
7. the method for observation microtissue inner cell solid distribution according to claim 5, which is characterized in that the closing
Treatment process are as follows: the permeable membrane liquid in the organization material of permeable membrane processing is removed, under conditions of 4~5 DEG C, by the tissue of permeable membrane processing
Material oscillation incubation 12~15 hours in confining liquid, the confining liquid ingredient include following quality component aqueous solution: 0.4~
0.6% Triton X-100,0.10~0.20% glycine, 9~11% fetal calf serum, 3~4% ox blood it is pure
Albumen.
8. the method for observation microtissue inner cell solid distribution according to claim 5, which is characterized in that the primary antibody
Being incubated for process with secondary antibody includes:
The organization material of Seal treatment is moved into primary antibody solution under the conditions of 4~5 DEG C by the confining liquid for S1) removing Seal treatment
In, it is incubated for 48~72 hours with the revolving speed shaking table of 50~60rpm；
S2 primary antibody solution) is removed, under the conditions of 2~5 DEG C, the Triton X-100 for being 0.1% with mass concentration is water-soluble
Liquid is cleaned, and is washed 3~4 hours with the revolving speed shaking table of 50~60rpm, and every the aqueous solution of replacement in 1 hour；
S3) the aqueous cleaning solution walked in removal moves into organization material in two corresponding anti-solution, under the conditions of being protected from light for 2~5 DEG C, with 50
The revolving speed shaking table of~60 rpm is incubated for 48~72 hours, if S1) in primary antibody be coupled with fluorophor, this step omission；
S4 two corresponding anti-solution) is removed, under the conditions of 42~5 DEG C, organization material is moved into the polyethylene glycol octyl of mass concentration 0.1%
Phenyl ether aqueous solution is cleaned, and is washed 3~4 hours with the revolving speed shaking table of 50~60rpm, and every the water of replacement in 1 hour
Solution, if S3) it omits, this step is omitted；
S5) the cleaning solution walked in removal moves into organization material in DAPI dilution under the conditions of being protected from light for 2~5 DEG C, with 50~
The revolving speed shaking table of 60 rpm is incubated for 10~12 hours, to identify that all cells in tagged tissue, the DAPI dilution are 4', 6-
Diamidino -2-phenylindone dilute aqueous solution.
9. the method for observation microtissue inner cell solid distribution according to claim 1, which is characterized in that described transparent
Changing processing step includes: to move into the organization material of immunofluorescence dyeing in transparent liquid, under the conditions of being protected from light for 20~25 DEG C, with 50
The revolving speed shaking table of~60 rpm is incubated for 48~72 hours, and the transparent liquid is the aqueous solution of following quality component: 22~26% urine
Element, 8~12% glycerol, 0.9~0.12% Triton X-100,48~52% sucrose.
10. a kind of method for counting microtissue inner cell number, which is characterized in that by according to any one of claims 1 to 9
The method for observing the distribution of microtissue inner cell solid obtains the cell distribution image of each layer of microtissue, passes through Imaris software
Each confluent monolayer cells distributed image data are handled, the threedimensional model of tissue is rebuild, are observed in three-dimensional stereo model by immunofluorescence label
The three-dimensional distribution of cell, recycles Imaris Bitplane Spot to test and analyze, calculates the standard of immunofluorescence label cell
Exact figures mesh.
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