CN107621462B

CN107621462B - Tissue clearing liquid SUT and preparation and application thereof

Info

Publication number: CN107621462B
Application number: CN201610548601.5A
Authority: CN
Inventors: 王志伟; 王巍; 张�杰; 范桄溥; 闫宇鹏
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-07-13
Filing date: 2016-07-13
Publication date: 2020-03-31
Anticipated expiration: 2036-07-13
Also published as: CN107621462A

Abstract

The embodiment of the invention relates to a tissue clearing liquid SUT which mainly comprises SDS, urea and TritonX-100. The invention also relates to the preparation and application of the tissue clearing liquid SUT. The tissue transparentizing liquid SUT provided by the embodiment of the invention reduces the cost and difficulty of preparation of the transparentizing liquid, increases the transparentizing effect, and has better transparentizing degree to cardiac muscle than a part of the conventional transparentizing liquid formula.

Description

Tissue clearing liquid SUT and preparation and application thereof

Technical Field

The invention relates to a tissue clearing liquid SUT and preparation and application thereof.

Background

Obtaining visual, complete tissue structure information at the single cell resolution level has been a long felt dream for scientists. By imaging the intact tissue with single cell resolution, a three-dimensional image of the intact tissue can be obtained, whereby the inter-cell connectivity and its dynamic changes can be elucidated at the organ and system level, which will greatly improve our understanding of the process of development of the disease, since single cell events in the disease will eventually lead to changes in the health status of the whole body. But the opacity of tissues and organs is the biggest obstacle to achieving this dream.

Classical protein qualitative and quantitative (namely determining which protein and the content of a certain protein) technologies comprise western blot, immunohistochemistry, immunofluorescence and the like, wherein (1) the western blot separates various proteins through an electrophoresis technology, can perform basic qualitative and quantitative analysis on the proteins, but cannot determine the spatial distribution position of the proteins in tissues and even cannot acquire three-dimensional images of protein distribution in the tissues; (2) because of the opacity of tissues and organs, traditional immunohistochemistry and immunofluorescence are performed on tissue sections (including paraffin sections and frozen sections), and the section thickness is less than 8 μm, which is beneficial to light transmission and fluorescent dye penetration. Wherein, the immunohistochemistry obtains a two-dimensional picture, and the three-dimensional distribution of the protein in the tissue can not be analyzed; immunofluorescence can be through hatching the fluorochrome to the tissue, then carry out three-dimensional reconstruction with confocal microscope, obtain the three-dimensional image of protein distribution, but the thickness of traditional immunofluorescence reconsitution is restricted by section thickness, the three-dimensional image thickness that obtains is too little (<8 μm), can't show the holomorphism to some great cells (such as left ventricle cardiac muscle cell, diameter 10 ~ 15 μm), and, if the tissue section is too thick, the fluorochrome (such as fluorescent antibody) can't pass through the tissue and carry out the protein mark, and confocal microscope's emission light also can't pass through the tissue whole layer, therefore three-dimensional reconstruction has very big limitation on using, at present mainly is used for taking fluorescence two-dimensional picture.

Because of the opacity of tissues and organs and the limitations of traditional research techniques, we cannot obtain the protein distribution in the whole tissues, and it is tried that if there is a method that can make the tissues have enough light transmittance, so that the emission light of confocal microscope can penetrate the whole tissue layer, and the fluorescent dye can also penetrate the whole tissue layer by performing specific treatment on the tissues, then the single cell resolution imaging of thicker whole tissues and the acquisition of the time-space distribution of proteins in the whole tissues can be realized, which will provide a new path for the mysterious secret revealing life, greatly improve our understanding of life, and at the same time, greatly improve our understanding of the disease occurrence and development process, and promote the health of human beings.

There are two factors that affect tissue light transmission: (1) light scattering-mainly due to differences in refractive index between layers of different tissue structures, where lipid composition influences most; (2) light absorption-mainly due to endogenous chromophore groups, of which hemoglobin and myoglobin predominate. There are two corresponding ways to increase tissue light transmission: (1) the difference of refractive indexes between different tissue structures is reduced as much as possible, and lipid is removed as much as possible; (2) blood and myoglobin were removed as much as possible from the tissues.

Tissue transparentization firstly fixes tissue protein through paraformaldehyde, and then forms a tissue gel compound by crosslinking with acrylamide, and the tissue gel compound has two characteristics: (1) the compound can fix protein and nucleic acid in situ, and can replace lipid to form a supporting structure for framing cells, so that the loss of protein can be reduced in the subsequent clearing process of the transparentizing liquid; (2) the tissue gel compound has different sizes and apertures, and fluorescent dye can enter the tissue through the small aperture and be combined with target protein, so that thicker tissue can be marked, and the defect of the traditional immunofluorescence is overcome. After a tissue gel compound is formed, the tissue is cleaned by a transparentizing liquid, lipid and endogenous chromophore are removed, so that the light transmittance of the tissue is greatly improved, and the visual information of the resolution level of the single cells of the complete tissue structure is obtained by incubating an antibody and a fluorescent dye to the transparent tissue, confocal imaging and 3D reconstruction.

Tissue transparentization requires that the maximum light transmittance is realized in the shortest time on the premise of keeping the original shape of the tissue as much as possible and minimizing the loss of protein, so that the real microstructure and the change information of the tissue are restored as much as possible. This method dates back to a century at the earliest, but the initial method caused a great disruption to the structure and morphology of the organization, and various methods were subsequently developed, including class, SCALE, SeeDB,3DISCO, CUBIC, PACT-PARS, etc., each of which contributed greatly to the secret of the people who uncovered life, and most of which were published in the journal of international authority. However, since various methods have been developed mainly for the study of the nervous system (brain tissue), 3DISCO has been used for other transparent organs, but it causes fluorescence quenching easily. The transparentizing method published in CELL journal in 2014 comprises CUBIC and PACT-PARS, wherein the transparentizing liquid of the CUBIC comprises two reagents which are required to sequentially treat tissues; the transparentizing liquid of the PACT-PARS is 8% SDS, the two transparentizing liquids are respectively perfused through a circulatory system to realize the whole body transparentization of mice, the PACT-PARS simultaneously carries out confocal scanning imaging of the brain, the structure of brain neurons is vividly displayed, but the research on other organs is superficial. The composition and structure of different tissues are different, so the transparentization conditions are slightly different, although CUBIC and PACT-PARS both realize the whole body transparentization of mice, the defects of the CUBIC and PACT-PARS are obvious:

(1) although CUBIC has better transparentization effect, the reagent composition is complex, and the reagent is not commonly used, wherein N, N, N ', N' -tetrahys (2-hydroxypropyl) Ethylene Diamine (EDTP) is very viscous, the solution preparation is difficult, the transparentization reagent comprises two types, the two types need to be sequentially used, the treatment time of each reagent is different, the operation steps are relatively complex, the EDTP is a copper ion settling agent, the conventional BCA method cannot be used for protein quantification, and simultaneously, the Nanodrop method for protein quantification is not accurate because CUBIC-1 is viscous;

(2) although simple, 8% SDS has poor clearing effect, severe tissue swelling, and high protein loss.

Disclosure of Invention

In order to solve the technical defects of poor transparentizing effect, high protein loss and the like of a transparentizing liquid in the prior art, the embodiment of the invention provides the following technical scheme.

One aspect of the present invention relates to a tissue clearing solution SUT, which is mainly composed of sds (sodium Dodecyl sulfate), Urea and triton x-100. Specifically, the concentrations of SDS, Urea and TritonX-100 in the tissue clearing solution SUT were as follows: SDS (wt/vol) is more than or equal to 4 percent and less than or equal to 12 percent, Urea (wt/vol) is more than 15 percent and less than or equal to 40 percent, Triton X-100(vol/vol) is more than or equal to 10 percent and less than or equal to 20 percent. Preferably, the concentrations of SDS, Urea and TritonX-100 in the tissue clearing fluid SUT are as follows: 8% SDS (wt/vol), 25% Urea (wt/vol), 15% TritonX-100 (vol/vol).

As another aspect of the present invention, the preparation method of the tissue clearing solution SUT described above is provided, wherein 4-12 g of solid SDS (sodium dodecyl sulfate) and 15-40 g of Urea are weighed into a glass bottle, a proper amount of 0.1M PBS (which may be replaced by distilled water or physiological saline, etc.) (less than 80ml) is added, the mixture is stirred and mixed uniformly, after the two are dissolved, liquid triton x-10010-20 ml is added, the mixture is heated and stirred uniformly, then 0.1M PBS (which may be replaced by distilled water or physiological saline, etc.) is added to a constant volume of 100ml, and the mixture is stirred uniformly.

As still another aspect of the present invention, the present invention relates to the use of the tissue clearing solution SUT described above for clearing tissue. In particular to the application of the tissue clearing liquid SUT in clearing heart tissues.

The tissue clearing liquid SUT provided by the embodiment of the invention at least has the following advantages:

the cost and difficulty of preparing the transparentizing liquid are reduced, the transparentizing effect is improved, and the transparentizing degree of the transparentizing liquid to cardiac muscle is superior to that of a part of the conventional transparentizing liquid formula (figure 2 a);

compared with the 8% SDS of the transparentizing liquid of PACT-PARS, the protein loss amount is less in the process of transparentizing the tissues (FIG. 2b), and the molecular structure of the tissues is retained to a greater extent;

while obtaining better tissue transparency, the original shape of the tissue is maintained as much as possible, the tissue volume swelling is reduced, and the tissue mass increase is reduced (fig. 2c and fig. 3);

the passive transparency of the whole left ventricle layer of a mouse (ICR/CD-1 mouse, 10 weeks) is realized in a short time (3-6 days), meanwhile, the whole heart transparency of the mouse (ICR/CD-1 mouse, 10 weeks) is completed only in 3 days, the passive transparentization of other organs of the mouse is successfully completed, a single-photon confocal microscope (single-photon confocal microscope) is used for obtaining a clear single-cell resolution 3D image of multiple organs of the mouse, and the molecular structure of each organ is presented in a living stereo manner.

Compared with the CUBIC transparentizing reagent combination, the cost and the difficulty of preparing the transparentizing liquid are reduced, the transparentizing process is simplified, the detection of the protein loss rate is facilitated, the transparentizing effect is improved, and the transparentizing degree of the transparent liquid to cardiac muscle is superior to that of the transparent liquid formula of CUBIC.

Compared with the 8% SDS of the transparentizing liquid of PACT-PARS, the protein loss amount is less in the process of tissue transparentizing, and the molecular structure of the tissue is retained to a greater extent; and the original form of the tissue is kept as much as possible while better tissue transparency is obtained, the tissue volume swelling is reduced, and the tissue mass increase is reduced.

Passive transparency of the whole left ventricle of a mouse (ICR/CD-1 mouse, 10 weeks) is realized in a short time (3-6 days), meanwhile, the whole heart of the mouse (ICR/CD-1 mouse, 10 weeks) is transparent only in 3 days, passive transparentization of other organs of the mouse is successfully completed, a single-photon confocal microscope (single-photon confocal microscope) is used for obtaining a clear single-cell resolution 3D image of multiple organs of the mouse, and the molecular structure of each organ is presented in a dynamic and three-dimensional manner.

The heart is a dense muscular organ, relatively low in fat content, high in hemoglobin and myoglobin, and different in hyalinization conditions from brain tissue. At present, the condition of transparentizing the cardiac muscle is not reported, and the embodiment of the invention provides a formula of a transparentizing liquid suitable for the cardiac muscle.

Drawings

FIG. 1: the subjects in FIG. 1 were 1.5mm thick heart cross sections of 10-week ICR/CD1 mice, 6 pieces per group, and cleared for 5 days with different concentrations of SDS after tissue gel complexes were formed. We selected 8% SDS as the reference and performed a one-way ANOVA analysis with SPSS (excluding PBS group), "-" indicating that this group was statistically different from the 8% SDS group.

Fig. 1(a) is a measurement result of the light transmittance of the treated tissue, and the 8% SDS group is not statistically different from the 4% SDS, 6% SDS, and 10% SDS groups, indicating that the 8% SDS, 4% SDS, 6% SDS, and 10% SDS have the same effect of clearing the tissue, while the 12% SDS, 16% SDS, and 20% SDS have the better light transmittance for the tissue, and it is considered that, only by comparing the light transmittances: the concentration of SDS is more than or equal to 4 percent;

FIG. 1(b) is a graph showing the measurement of the protein loss rate, which is statistically different in each of the other groups compared with the 8% SDS group, and shows that the protein loss rate after the tissue was treated with 8% SDS is consistent with the protein loss rate after the tissue was treated with SDS at each of the other concentrations;

FIG. 1(c) is a measurement of the percentage of tissue mass increase, which is statistically different in the 10% SDS group compared to the 8% SDS group, and the percentage of tissue mass increase is less than 8% SDS, closer to the initial state of the tissue;

we think that the light transmittance of tissues treated by SDS is low, and practice of liquid preparation finds that when the concentration of SDS exceeds 12%, the addition of other transparentizing agents is not facilitated, so that the concentration range of SDS is set to be between 4% and 12% in combination with the above experimental results.

FIG. 2: the study subjects in FIG. 2 were 10-week ICR/CD1 mice, 1.5mm thick heart cross sections, 6 pieces per group, and after tissue gel complexes were formed, they were cleared for 5 days with solutions of varying formulation. Wherein S-8% SDS, SUx-8% SDS + X% Urea, SUTy-8% SDS + 25% Urea + y% triton X-100;

(1) in the S-SU 40 group, we selected SU25 as the reference subject and performed one-way ANOVA analysis using SPSS, ". X" indicates that the group is statistically different from SU 25.

FIG. 2(a) is a measurement of light transmittance, which is statistically different from that of SU25 group when the Urea concentration is increased from 0 to 15% (SU 25 group)₁₅vs SU₂₅P ═ 0.029), when the Urea concentration increases to 20%, and SU₂₅Group comparisons were not statistically different (SU)₂₀vs SU₂₅P ═ 0.066), when Urea concentration increased to 30% and 35%, and SU₂₅No statistical difference was found in group comparisons (SU)₂₅vs SU₃₀，p＝0.445；SU₂₅vs SU₃₅P ═ 0.054), when the Urea concentration increased to 40%, and SU₂₅Group comparisons were statistically different (SU)₂₅vs SU₄₀P is less than 0.001), which shows that the Urea concentration is less than or equal to 35 percent in the case of mixing Urea and 8 percent SDS solution, the transparentizing effect on the tissues is consistent, and is better than that when the Urea concentration is less than or equal to 15 percent and is increased to 40 percent, the transparentizing effect on the tissues is better, so that the optimal concentration range of the Urea in the range of 15 percent to less than or equal to 40 percent is determined; FIG. 2(b) is a measurement result of protein loss rate, S to SU₁₅Group sum SU₂₅All group comparisons were statistically different (SU)₁₅vs SU₂₅P is 0.028), all other groups were statistically different, indicating that S to SU₁₅The protein loss rate after tissue treatment is obviously higher than that of SU₂₅Group, SU₂₀～SU₄₀Processing group and SU₂₅The loss rate of the protein of the group phase is consistent, so that the optimal concentration range of Urea is more than 15 percent from the aspect of the protein loss rate; FIG. 2(c) is a measurement of percent tissue mass growth, S-SU₄₀Group and SU₂₅None of the group comparisons were statistically different. In addition, in the process of liquid preparation, when the Urea and 8% SDS are mixed, when the Urea concentration is higher than 40%, the addition of other transparentizing reagents is not facilitated, and the solution is unstable at low temperature, and in conclusion, the optimal concentration range of Urea from 15% to 40% is determined;

(2)SUT₅～SUT₂₀SeeDB, Scale A2, CUBIC-1 group, we selected CUBIC-1 group as the reference and used SPSS to perform one-way ANOVA analysis, and ". x" indicates that the group is statistically different from CUBIC-1 group.

FIG. 2(a) is a measured value of light transmittance, CUBIC is an authoritative method published in the journal CELL, and we believe that the CUBIC-1 agent has a better effect of transparentizing the myocardium, so we use the CUBIC-1 treatment group as a reference group of light transmittance and obtain SUT through statistical analysis₅Has significant statistical difference (P is less than 0.001) with the CUBIC-1 group, namely the transparentization effect of CUBIC-1 on tissues is far more than that of SUT₅，SUT₁₅The group and CUBIC-1 group have significant statistical difference (P < 0.001), namely SUT15 has much more transparentizing effect on tissues than CUBIC-1, while SUT₁₀And SUT₂₀No statistical difference (SUT) was observed between the group and the CUBIC-1 group₁₀vs CUBIC-1，p＝0.067；SUT₂₀vs CUBIC-1, p ═ 0.169), stating SUT₁₀、SUT₂₀The effect of clearing the myocardial tissue was comparable to CUBIC-1. Therefore, by comparing the light transmittances, the optimal concentration range of TritonX-100 of more than or equal to 10 percent is determined to be less than or equal to 20 percent; FIG. 2(b) is a measurement of the protein loss rate, SUT₅、SUT₁₀、SUT₂₀And SUT₁₅In contrast, all were statistically different (SUT)₅vs SUT₁₅，p＝0.004；SUT₁₀vs SUT₁₅，p＝0.012；SUT₂₀vs SUT₁₅P is 0.004), fig. 2(c) is a tissue mass growth percentage measurement, SUT₅、SUT₁₀、SUT₂₀And SUT₁₅In contrast, all were statistically different (SU)_T5vs SUT₁₅，p＜0.001；SUT₁₀vs SUT₁₅，p＝0.015；SUT₂₀vs SUT₁₅P < 0.001). According to practice, we found that SUT can be used₁₀And SUT₂₀The treated tissue can be subjected to subsequent incubation of fluorescent dye and copolymerization set microscopic imaging, and the effect is superior to that of 8% SDS (sodium dodecyl sulfate) in a clearing solution published in an authoritative journal CELL, so that the optimal concentration range of TritonX-100 is more than or equal to 10% and less than or equal to 20% by the final determination.

FIG. 3: the study object in fig. 3 is a 10-week ICR/CD1 mouse left ventricle full-thickness circular slice (cut with a punch, diameter 4.5mm), each group has 10 slices, after forming a tissue gel complex, two groups are respectively cleared with PACT-PARS clearing solution 8% SDS and our clearing solution SUT (the ratio of 8% SDS + 25% Urea + 15% triton x-100 taken this time), then the change in tissue volume before and after clearing is calculated with Imagej, and an independent sample t test is performed with SPSS, to obtain that two groups have a significant statistical difference (p < 0.001), which indicates that compared with 8% SDS, the change in tissue volume before and after clearing of SUT is smaller and closer to the original form of the tissue.

The specific implementation mode is as follows:

the present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.

The preparation method of the tissue clearing liquid SUT provided by the embodiment of the invention comprises the following steps:

weighing 4-12 g of solid SDS (sodium dodecyl sulfate) and 15-40 g of Urea (Urea) in a glass bottle, adding a proper amount of 0.1M PBS (which can be replaced by distilled water or normal saline and is less than 80ml), uniformly stirring, dissolving, adding liquid TritonX-10010-20 ml, heating and uniformly stirring, then adding 0.1M PBS (which can be replaced by distilled water or normal saline and the like) to fix the volume to 100ml, and uniformly stirring.

The tissue clearing liquid SUT provided by the embodiment of the invention comprises the following components:

the solute mainly comprises SDS, Urea and TritonX-100, and the solvent is 0.1M PBS (or distilled water or physiological saline can be used for replacing).

We chose the documents Yang B, Treweek J B, Kulkarni R P, et.Single-Cell phenotyping with transpatient interaction tissue through hole-body clearing [ J ] Cell,2014,158(4):945 958 and Chung K, deisseoth K.Clarity formatting the nervous system [ J ] Nature methods,2013,10(6):508 513. for reference, use the myocardium as the object of clearing, rescreen the optimal concentration of SDS, by adding a variety of possible clearing agents including tetrahydroxypropylethylenediamine (EDD), D-Fructose, Urea, Glycerol, TritonX-100, finally determine the thickness of the tissue to which Urea and TritonX-100 are added, and search for the optimal thickness of each set of slides as the cross-section, and search for the cross-section of the tissue by the method of 1. 5. for each set of slides, the transparentization was carried out for 5 days with different combinations of transparentizing liquids. The screened target clearing liquid is to reduce the protein loss rate and the change of the original form of the tissue as much as possible on the premise of ensuring enough light transmittance.

EDTP (5%, 10%, 15%, 20%, 25%, 30%, 35%) of each concentration gradient was mixed with 8% SDS, and after 5 days of transparency, we found that the tissue yellowing was significant and the protein loss rate could not be determined with the conventional BCA kit, so it was discarded; D-Fructose (10%, 20%, 40%, 60% and 80%) with each concentration gradient is mixed with 8% SDS respectively, and the solution preparation finds that the D-Fructose is difficult to dissolve with the 8% SDS, and when the concentration is higher than 60%, the solution is unstable, and the mixed solution of the D-Fructose and the SDS with each concentration has poor tissue transparentization effect, so the solution is abandoned; mixing glyceol (5%, 10% and 20%) with each concentration gradient with 8% SDS respectively, and after 5 days of transparency, finding that the mixed solution of glyceol and SDS with each concentration has poor effect on transparentizing tissues, so that the mixed solution is abandoned; when Urea and 8% SDS with various concentrations are mixed, the tissue transparentizing effect is obviously increased, and the tissue transparentizing effect is better after TritonX-100 is added, so that the addition of Urea and TritonX-100 is finally determined.

The experimental steps are as follows:

1. deeply anaesthetizing 10-week ICR/CD1 mice by intraperitoneal injection of 0.7ml of 3% chloral hydrate, and fixing limbs;

2. after the abdominal operation, performing inferior vena cava puncture, cutting the abdominal aorta, and sequentially perfusing PBS Flush 40ml and 4% Paraformelhyde 20ml from the inferior vena cava;

3. opening the chest to core, cutting a transverse section of the heart with the thickness of 1.5mm by using a self-made row knife with the interval of 1.5mm, cutting 3 slices of each heart and 6 slices of each experimental group (for measuring the volume of the tissue conveniently, a circular puncher with the diameter of 4.5mm is used for cutting a circular section with the diameter of 4.5mm on the whole layer of the left chamber wall, 1 slice of each heart and 10 slices of each of the two experimental groups, and the edge of the circular section passes through the root parts of the front and rear papillary muscles);

4. gently wiping each tissue dry, weighing, photographing, and measuring the light transmittance;

5. placing each section in 4% Paraformedehyde, and standing overnight at 4 deg.C;

6. each section was transferred from 4% paraformethylene to 4% Acrylamide containing 0.25% VA044 overnight at 4 ℃;

7. the tubes soaked with tissue sections, 4% Acrylamide (containing 0.25% VA044), were removed from 4 deg.C, sequentially purged with nitrogen on ice for 2 minutes per tube, and then placed in a 37 deg.C water bath for 2-3 hours;

8. preparing transparentizing liquid with various concentration ratios, subpackaging into 50ml centrifuge tubes, transferring the tissues into various transparentizing liquids with corresponding labels after water bath (the transparentizing liquids are 2-20% SDS and SU respectively₅～SU₄₀、SUT₅～SUT₂₀、SeeDB、ScaleA₂CUBIC-1, wherein SUx 8% SDS + X% Urea, SUTy 8% SDS + 25% Urea + y% Triton X-100, SeeDB ═ free (80.2% wt/wt, in water) + 0.5% α -thioglycerol, ScaleA2 ═ 4M Urea + 10% (wt/vol) glycerol + 0.1% (wt/vol) Triton X-100, CUBIC-1-free (25 wt% Urea +25 wt% N, N, N ', N' -tetrapropyl (2-xypropyl) ethylene diamine +15 wt% polyethylene glycol mono-p-iso ethylene ether/triton X-100), and a liquid containing 50ml of each of the above-mentioned transparent microcentrifuge and the obtained mixturePlacing into a shaking table at 37 ℃, shaking and incubating for 5 days, taking out tissues, and measuring and calculating corresponding parameters (mass growth percentage, protein loss rate, tissue light transmittance and volume growth percentage).

The mass is weighed by a high-precision electronic balance, the precision is 0.001g, and the mass increase percentage (weight increase%) is equal to (mass of transparent rear tissue-mass of transparent front tissue)/mass of transparent front tissue; protein loss rate was determined by BCA assay, BCA protein quantification kit (P0010) was purchased from Beyotime, protein loss rate is total protein amount in clearing solution/tissue mass before clearing, i.e. total protein loss/weight of uncleared tissue; measuring the light transmittance of a transverse slice with the thickness of 1.5mm by using an optical transmittance measuring instrument (LS108D), randomly measuring 6 positions of each slice, and averaging; percent volume increase was measured by imagej software after photographing with CANON 700D, percent volume increase is (volume of transparent posterior tissue-volume of transparent anterior tissue)/volume of transparent anterior tissue.

The experimental results are as follows:

(1) in order to screen an optimal SDS concentration range, a series of SDS concentration gradients are set, and by comparing weight increase percentage, total protein loss/weight of unclean concentration and light transmittance, and combining solution preparation practice, an optimal concentration range (wt/vol) of 4% to 12% of SDS is obtained (figure 1);

(2) according to the documents Hama H, Kurokawa H, Kawano H, et al, Scale: a chemical improvement for fluorescence imaging and recovery of transient mouse blue [ J ]. Nature neuroscience,2011,14(11): 1481:. 1488. when mixed with 8% SDS, ① Urea concentration is between 15% and 40%, the tissue transparency is good and the solution is stable, ② when Urea concentration is higher than 40%, the solution is concentrated and not beneficial to adding other transparentizing agents, ③ when Urea concentration is increased to higher than 45%, the solution becomes unstable and is prone to crystal precipitation at lower temperature (lower than 20%), and when comprehensive consideration is given, the range of 15 Urea concentration is selected (Urea concentration/wt) < 2% (FIG. 2/vol.);

(3) with the addition of TritonX-100, the tissue transparency is better and better, and the transparentization effect is optimal when the concentration range of TritonX-100 is 10% -20%; protein BCA quantification is carried out on each group of clearing solutions after 5 days of clearing, and the result shows that the loss amount of the SUT15 and SUT20 histones is minimum; we believe that the tissue transparency is better after the CUBIC-1 treatment, so we refer to the CUBIC-1 treatment group as a reference in the comparison of transparency. By integrating the transparentization effect, the protein loss rate and the mass growth percentage, we think that the optimal concentration range (vol/vol) of TritonX-100 is more than or equal to 10 percent and less than or equal to 20 percent (figure 2).

According to the above results, the solute of SUT comprises three components, SDS (sodium dodecyl sulfate), Urea (Urea), Triton X-100, and the concentration ranges of each agent are: the concentration of SDS (wt/vol) is more than or equal to 4 percent and less than or equal to 12 percent, the concentration of Urea (wt/vol) is more than 15 percent and less than or equal to 40 percent, and the concentration of TritonX-100(vol/vol) is more than or equal to 10 percent and less than or equal to 20 percent; the solvent was 0.1M PBS (distilled water or physiological saline may be used instead of it).

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention.

Claims

1. The SUT is characterized by mainly comprising SDS, urea and TritonX-100, wherein the concentration dosage of the SDS, the urea and the TritonX-100 is as follows: SDS is between 4 percent and 12 percent, and calculated by wt/vol; 15 percent < urea less than or equal to 40 percent in wt/vol; TritonX-100 is more than or equal to 10 percent and less than or equal to 20 percent in terms of vol/vol.

2. The SUT according to claim 1, wherein the concentrations of SDS, Urea and TritonX-100 are as follows: 8% SDS in wt/vol; 20-30% urea, in wt/vol; 15% TritonX-100, in vol/vol.

3. The SUT according to claim 2, wherein the concentrations of SDS, Urea and TritonX-100 are as follows: 8% SDS in wt/vol; 25% urea, in wt/vol; 15% TritonX-100, in vol/vol.

4. The SUT according to claim 3, wherein said SUT further comprises a pH adjusting agent and/or an osmotic pressure adjusting agent.

5. A method for preparing the SUT according to any of claims 1 to 4, wherein the method comprises: weighing 4-12 g of solid SDS and 15-40 g of urea, adding a proper amount of 0.1M PBS, distilled water or normal saline into a glass bottle, uniformly stirring, adding 10-20 ml of liquid TritonX-100 after the two are dissolved, heating and uniformly stirring, then fixing the volume to 100ml by using 0.1MPBS, distilled water or normal saline, and uniformly stirring.

6. Use of the tissue clearing fluid SUT according to any one of claims 1-4 for clearing tissue.

7. The use according to claim 6, wherein said use is in the clearing of cardiac tissue.