Anti-reflection method and reagent suitable for rapid marking of complete organ cell construction

Abstract

The invention belongs to the technical field of biological tissue sample dyeing and three-dimensional imaging, and particularly relates to an anti-reflection method and reagent suitable for rapid marking of complete organ cell construction. The anti-reflection dyeing reagent provided by the invention comprises a composite anti-reflection agent, a dyeing agent and a solvent, and the composite anti-reflection agent comprises a detergent, a super-hydrating agent and a cholesterol remover, so that a cell nucleus dye and a biomacromolecule fluorescence coupling marker can effectively mark a complete organ; and the staining time of the complete mouse brain cell construction can be shortened from 3-4 weeks to 5 days in a passive soaking manner. According to the anti-reflection method based on the reagent, complete organ cell construction marking can be rapidly achieved in a high-quality mode only through a passive soaking mode, and the method has the advantages of being easy and convenient to operate, high in universality, short in dyeing time, high in uniformity, high in signal-to-background ratio and the like; the method is suitable for three-dimensional reconstruction space positioning, cell counting and morphological research in physiological and pathological research, accurate anatomical atlas drawing and the like.

Description

Technical Field

[0001] This application belongs to the field of biological tissue sample staining and three-dimensional imaging technology, and more specifically, relates to an enhanced transparency method and reagent suitable for rapid labeling of intact organ cell structures. Background Technology

[0002] In life science research, in addition to specifically fluorescently labeled target structures, three-dimensional localization studies are also required within the spatial anatomical annotation of intact organs. This necessitates simultaneously acquiring cellular architecture information of the complete organ for spatial localization, identification of anatomical regions, and establishment of a spatial coordinate system. Therefore, fluorescent labeling of cellular architecture in biological tissues is necessary.

[0003] Currently, the main cell construction labeling strategies for intact organs include passive immersion and real-time counterstaining. Real-time counterstaining alternates between cutting and imaging, allowing staining to be performed simultaneously with sample cutting. It can utilize the tens of seconds interval between cutting and imaging to complete the staining of surface tissues, avoiding the time overhead of independent staining. However, it is generally used in optical imaging methods for intact organs that combine imaging and cutting. Moreover, within a limited time window, the penetration depth of commonly used nuclear fluorescent staining agents (such as DAPI, PI, Hoechst 33342, etc.) is limited, generally within ten micrometers. Passive immersion is simple to operate and has good versatility, but when staining intact organs, the penetration rate of the staining agent is slow, and there is a large difference in signal intensity between the organ surface and the center. For example, staining an intact mouse brain requires about 3 to 4 weeks.

[0004] Therefore, there is an urgent need in the field for an enhanced staining reagent that can rapidly and with high quality achieve markers of intact organ cell construction through passive immersion alone. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this application is to provide a permeation-enhancing method and reagent suitable for rapid labeling of intact organ cell construction. This aims to solve the problems of slow staining speed and limited penetration depth leading to long staining cycles, large differences in staining signal intensity between the organ surface and the center, and poor staining uniformity when using existing permeation-enhancing staining agents to label the construction of intact organs.

[0006] To achieve the above objectives, in a first aspect, this application provides a permeation-enhancing staining reagent suitable for rapid labeling of intact organ cells, comprising a composite permeation enhancer, a staining agent, and a solvent; The aforementioned composite permeability enhancer is composed of detergent, superhydrating agent and cholesterol removal agent, and is used to improve the permeability of intact organs to the staining agent.

[0007] Preferably, the detergent is one or more of CHAPS, Triton X-100, Tween 20, saponins, and SDS.

[0008] Preferably, the superhydrating agent is one or more of urea, formamide, 1,3-cyclohexanedimethylamine, and m-phenylenediamine.

[0009] Preferably, the cholesterol removal agent is one or more of β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, cholesterol oxidase, digitoxin, and fenipin.

[0010] Preferably, when the concentration of the detergent in the above-mentioned brightening dyeing reagent is 0.1% to 1% (w / v), the concentrations of the superhydrating agent and the cholesterol removal agent are each independently 1% to 20% (w / v), and the total concentration of the superhydrating agent and the cholesterol removal agent is greater than 2% (w / v).

[0011] Preferably, when the concentration of the detergent in the above-mentioned transparent dyeing reagent is greater than 1% (w / v), the concentrations of the superhydrating agent and the cholesterol removal agent are each independently 0.1% to 20% (w / v), and the total concentration of the superhydrating agent and the cholesterol removal agent is greater than 0.2% (w / v).

[0012] More preferably, the concentration of the detergent in the above-mentioned brightening dyeing reagent is greater than 1% (w / v) and less than 20% (w / v).

[0013] Preferably, the staining agent is one or more of the following: nuclear fluorescent markers, protein markers, fluorescent Nissl dyes, and lectin fluorescent conjugates.

[0014] Preferably, the concentration of the dye in the above-mentioned brightening dyeing reagent is 1 μg / mL to 200 μg / mL.

[0015] Preferably, the solvent is deionized water, phosphate buffer, cell culture buffer, or a combination thereof.

[0016] Secondly, this application provides a permeation enhancement method suitable for rapid labeling of intact organ cells, comprising the following steps: The pretreated intact organ was immersed in the above-mentioned transparent staining reagent to achieve the marking of intact organ cell construction.

[0017] Preferably, the above pretreatment methods include immunostaining, chemical dye staining, viral labeling staining, or transgenic staining of intact organs.

[0018] Preferably, the above-mentioned anti-reflection method further includes three-dimensional imaging of the complete organ with cellular construction markers, wherein the three-dimensional imaging includes the following steps: The complete organ with the cell construction markers described above was embedded, and then the embedded complete organ was imaged; The imaging method includes fluorescent microscopy optical sectioning tomography, optical transparent tissue light sheet imaging, or two-photon sequential tomography.

[0019] In a third aspect, the present application provides a use of the above-mentioned penetration enhancing staining reagent in three-dimensional reconstruction space positioning, physiological and pathological research cell counting and morphology research, and precise anatomical atlas drawing.

[0020] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) The penetration enhancing staining reagent provided by the present application is suitable for cell construction marking of complete organs, which comprises a composite penetration enhancer, a staining agent and a solvent. The composite penetration enhancer is compounded by a detergent, a super-hydration agent and a cholesterol removing agent. Through the synergistic effect between the components, the penetration of the staining agent into the complete organ can be significantly improved, the staining speed of the complete organ can be significantly improved, and the staining uniformity can be effectively improved. The complete organ cell construction marking can be quickly and high-quality realized by passive soaking.

[0021] (2) By compounding suitable types of detergent, super-hydration agent and cholesterol removing agent and optimizing the concentration of each component, the staining depth of the staining agent on large-size biological tissue samples can be significantly improved while reducing the staining cost. The staining depth effect is significantly better than that of a single penetration enhancer component at the same concentration.

[0022] (3) Compared with the existing penetration enhancing staining agent, the composite penetration enhancer in the penetration enhancing staining reagent provided by the present application can be compatible with various cell construction dyes to realize different specific fluorescent staining schemes, and can also be adapted to different sizes of complete biological tissues to realize rapid staining of complete biological tissues without damaging the biological tissues. It also does not affect other fluorescent markers, ensures that the staining effect after pretreatment of different staining schemes can be well maintained after penetration enhancing staining, has the advantages of simple operation, strong universality, low staining cost, short staining time, strong uniformity, high signal-to-background ratio, and is suitable for three-dimensional reconstruction space positioning, physiological and pathological research cell counting and morphology research, and precise anatomical atlas drawing. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a maximum coronal plane cell construction image of a complete mouse brain treated by the penetration enhancing staining reagent provided by the embodiment and obtained by a fluorescent microscopy optical sectioning tomography system; Figure 2 is a maximum coronal plane cell construction image of a complete mouse brain treated by a PI solution and obtained by a fluorescent microscopy optical sectioning tomography system. DETAILED DESCRIPTION

[0024] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0025] In the description of the present application, it should be understood that the term "and / or" is a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. In this paper, the symbol " / " represents the relationship of or, for example, A / B represents A or B.

[0026] In the description of the embodiments of the present application, the words "exemplary" or "for example" are used to represent an example, illustration or description. Any embodiment or design scheme described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as more preferred or more advantageous than other embodiments or design schemes. On the contrary, the words "exemplary" or "for example" are intended to present the relevant concept in a specific way.

[0027] In the description of the embodiments of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0028] The present application provides a transmittance dyeing reagent suitable for cell construction marking of intact organs, which comprises a composite transmittance agent, a dyeing agent and a solvent. The composite transmittance agent is compounded by a detergent, a super-hydrating agent and a cholesterol removing agent, and the composite transmittance agent is used to improve the permeability of the intact organs to the dyeing agent.

[0029] In some embodiments, the detergent is one or more of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid), Triton X-100, Tween 20, saponin, SDS; the super-hydrating agent is one or more of urea, formamide, 1,3-cyclohexanedimethylamine, m-xylylenediamine; and the cholesterol removing agent is one or more of β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, cholesterol oxidase, digitonin, filipin.

[0030] The application can significantly improve the dyeing depth of the dyeing agent on the intact organ by compounding suitable kinds of detergents, super-hydrating agents and cholesterol removing agents. The analysis reason may be that the detergent in the composite penetration enhancer has hydrophilic group and hydrophobic group at both ends of the molecule, which can self-assemble into micelles in water, wrap hydrophobic molecules inside, form micelles with cell membrane phospholipids and form holes on the membrane, effectively improve the cell membrane permeability; at the same time, the cholesterol removing agent has a high affinity with the cholesterol in the cell membrane due to its cyclic hydrophobic cavity, which can remove a large amount of membrane cholesterol, reduce the stability of the membrane structure, enhance the permeability, and more easily form micelles with the detergent, further improve the membrane permeability. In addition, the super-hydrating agent containing a large number of amino groups has a stronger hydrogen bonding ability than water, which can competitively replace the water between macromolecular proteins, cause the disintegration of collagen fibers, cytoskeleton and other high-level structures, and affect the arrangement of membrane phospholipids, thereby improving the cell membrane permeability. Through the synergistic penetration effect between the components in the composite penetration enhancer, the penetration and dyeing reagent prepared by mixing the above composite penetration enhancer with the dyeing agent can effectively improve the permeability of macromolecular dyeing agent to intact biological tissues when the cell structure of the intact organ is dyed, thereby significantly improving the dyeing speed of the intact biological tissue, and effectively improving the dyeing uniformity.

[0031] In some embodiments, when the concentration of the detergent in the above-mentioned penetration and staining reagent is 0.1% to 1% (w / v), the concentration of the super-hydration agent and the concentration of the cholesterol removing agent are each independently 1% to 20% (w / v), and the sum of the concentration of the super-hydration agent and the concentration of the cholesterol removing agent is greater than 2% (w / v), the penetration and staining reagent significantly improves the staining depth, and the staining effect of the single penetration component at the same concentration is significantly better than that of the single penetration component at the same concentration, which can effectively improve the staining depth of the staining agent on the intact biological tissue at a low cost. In some embodiments, the concentration of the detergent in the above-mentioned penetration and staining reagent can be 0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6% (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (w / v), 1% (w / v), and the like. In some embodiments, the concentration of the super-hydration agent and the concentration of the cholesterol removing agent can each independently be 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 9% (w / v), 10% (w / v), 11% (w / v), 12% (w / v), 13% (w / v), 14% (w / v), 15% (w / v), 16% (w / v), 17% (w / v), 18% (w / v), 19% (w / v), 20% (w / v), and the like. In some embodiments, the sum of the concentration of the super-hydration agent and the concentration of the cholesterol removing agent can be 3% (w / v), 5% (w / v), 8% (w / v), 10% (w / v), 15% (w / v), 18% (w / v), 20% (w / v), and the like. In preferred embodiments, the sum of the concentration of the super-hydration agent and the concentration of the cholesterol removing agent is greater than 10% (w / v).

[0032] In some embodiments, when the concentration of the detergent in the above-mentioned penetration and staining reagent is greater than 1% (w / v), the concentration of the super-hydration agent and the concentration of the cholesterol removing agent are each independently 0.1% to 20% (w / v), and the sum of the concentration of the super-hydration agent and the concentration of the cholesterol removing agent is greater than 0.2% (w / v), the penetration and staining reagent significantly improves the staining depth of the intact organ, which is significantly better than that of CHAPS with a mass-volume concentration of 20%, and can greatly improve the staining effect while reducing the cost of the cell structure staining of the intact organ.

[0033] In preferred embodiments, the concentration of the detergent in the above-mentioned penetration and staining reagent is greater than 1% (w / v) and less than 20% (w / v). For example, the concentration of the detergent can be 5% (w / v), 10% (w / v), 15% (w / v), and the like.

[0034] In a better embodiment, the concentration of the detergent in the above-mentioned transmittance-increasing and staining reagent is 10% (w / v), the concentration of the superhydration agent is 0.1%-20% (w / v) (specifically, 0.1% (w / v), 0.3% (w / v), 0.5% (w / v), 1% (w / v), 3% (w / v), 5% (w / v), 10% (w / v), 15% (w / v), 18% (w / v), 20% (w / v), etc.), the concentration of the cholesterol removing agent is 0.1%-10% (w / v) (specifically, 0.1% (w / v), 0.3% (w / v), 0.5% (w / v), 1% (w / v), 3% (w / v), 5% (w / v), 8% (w / v), 10% (w / v)), and the total concentration of the superhydration agent and the cholesterol removing agent is greater than 0.2% (w / v). When the complete organ cell construction labeling is performed by using the transmittance-increasing and staining reagent, the complete biological tissue cell construction labeling can be quickly and high-quality completed by only the passive soaking method, the staining time of the complete organ is obviously shortened, and the staining uniformity is excellent.

[0035] The composite transmittance-increasing agent in the transmittance-increasing and staining reagent provided in the present application can be compatible with various dyes to realize different specific staining schemes. In some embodiments, the above-mentioned staining agent includes but is not limited to one or more of a fluorescent nuclear marker (for example but not limited to 4', 6-diamino-2-phenylindole (DAPI), propidium iodide (PI), Hoechst 33342, SYTO 16), a protein marker (for example but not limited to rhodamine, Alexa Fluor series dye combined with NHS ester, etc.), a fluorescent Nissl dye (for example but not limited to NeuroTrace TM series, etc.), a lectin fluorescent conjugate (for example but not limited to LEL-Dylight488, etc.).

[0036] The transmittance-increasing and staining reagent provided in the present application can be adapted to different sizes of complete biological tissues by adjusting the concentration of the staining agent, realize the rapid staining of the complete biological tissue and avoid the damage to the biological tissue. It can be understood that the concentration of the staining agent in the above-mentioned reagent is not specially limited in the present application, and in actual use, the person skilled in the art can select the appropriate concentration according to the size of the biological tissue and the type of the staining agent. In some embodiments, the concentration of the staining agent in the above-mentioned reagent can be 1-200 μg / mL. In some embodiments, when the above-mentioned staining agent is propidium iodide, the concentration of propidium iodide in the above-mentioned reagent can be 30-100 μg / mL.

[0037] It can be understood that the application does not have special limitations on the solvents in the above-mentioned penetration dyeing reagent, and the solvents commonly used in the art are suitable for the application. In some embodiments, the above-mentioned solvent can be, but is not limited to, deionized water, phosphate buffer solution (PBS), cell culture buffer (such as but not limited to HEPES), or a combination of any of the above.

[0038] In another aspect, the application also provides a penetration method suitable for rapid labeling of cell architecture of a whole organ, comprising the following steps: Soaking the pretreated whole organ in the above-mentioned penetration dyeing reagent to realize cell architecture labeling.

[0039] In some embodiments, the above-mentioned pretreatment method comprises immunostaining, chemical dye staining, virus marker staining or transgenic staining of the whole organ.

[0040] In some embodiments, the above-mentioned method further comprises three-dimensional imaging of the whole organ on which the cell architecture labeling is realized, and the three-dimensional imaging comprises the following steps: Embedding the whole organ on which the cell architecture labeling is realized, and then imaging the embedded whole organ; The imaging method comprises fluorescence microscopic optical section tomography, optical transparent tissue optical section imaging or two-photon sequential tomography.

[0041] By compounding suitable types of detergents, super-hydrating agents and cholesterol-removing agents and adjusting the suitable concentrations of the components, the penetration dyeing reagent prepared by the application can significantly improve the staining depth of the dyeing agent on the whole organ, and can quickly and high-quality complete the cell architecture labeling of the whole biological tissue by passive soaking, has the advantages of simple operation, strong universality, low dyeing cost, short dyeing time, excellent dyeing uniformity, etc., and is suitable for three-dimensional reconstruction spatial positioning, cell counting and morphological research in physiological and pathological research, and drawing accurate anatomical atlas. Based on this, the application also protects the application of the above-mentioned penetration dyeing reagent in three-dimensional reconstruction spatial positioning, cell counting and morphological research in physiological and pathological research, and drawing accurate anatomical atlas.

[0042] The whole organ in the application refers to a clinical tissue or an animal organ after being removed from the body. The animal can be an experimental animal commonly used in the field of life science research, such as a mouse, a rabbit, a pig, a cow, a sheep, etc. The penetration dyeing reagent and the penetration method provided by the application are used for scientific research, not for diagnostic detection or auxiliary detection.

[0043] It should be understood that the same or similar materials in terms of type, model, quality, property or function of reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0044] The following are examples and comparative examples: Examples 1-17 (1) Pretreatment Take one two-month-old C57 mouse, pre-label the target organ specifically by tail vein injection, and after the intravascular fluorescent marker is fully circulated and labeled, anesthetize the mouse by intraperitoneal injection of a mixture of 10% urethane and 2% chloral hydrate, then open the chest to expose the heart, cut the right auricle and insert the needle into the left ventricle from the apex, perfuse 37°C PBS solution at a rate of about 15 mL / min for 3-5 min, and observe that the liver turns white, then perfuse 4°C 4% PFA solution at the same rate for 5 min, and observe that the mouse limbs and tail are rigid, indicating that the perfusion is sufficient. Take out the intact target organ (mouse brain) and fix it in 4°C 4% PFA solution for 12 h to obtain the pretreated intact organ.

[0045] (2) Cell construction staining Place the fixed intact mouse brain (length, width and thickness dimensions of 15 mm x 10 mm x 6 mm) in the penetration-enhancing staining reagent and stain at room temperature. The penetration-enhancing staining reagent provided by Comparative Examples 1-3 of the present application is obtained by mixing a single penetration enhancer (CHAPS, urea or 2,6-dimethyl-β-cyclodextrin) with a staining agent (propidium iodide). The penetration-enhancing staining reagent provided by Examples 1-17 and Comparative Examples of the present application is obtained by mixing a composite penetration enhancer (including CHAPS, urea and 2,6-dimethyl-β-cyclodextrin) with a staining agent. The concentration of the staining agent (propidium iodide) in the above-mentioned penetration-enhancing staining reagent is 20 μg / mL; the mass / volume concentration of the penetration enhancer is shown in Tables 1-4.

[0046] After staining for 90 min, calculate the average fluorescence signal-to-background ratio of the stained area at the periphery of the brain slice, then find the cells whose signal-to-background ratio decreases to 70% of the periphery staining area along the direction perpendicular to the edge of the brain slice, and the geometric center of the cells furthest from the edge meets the above requirements as the boundary of the staining area. The distance from the boundary to the edge of the brain slice is the staining depth, and the results are shown in Tables 1-4.

[0047]

[0048]

[0049]

[0050]

[0051] As can be seen from the dyeing depth data in Tables 1-3, the dyeing reagent prepared by using only one kind of penetrating agent alone with the dyeing agent cannot effectively improve the dyeing degree of the intact organ, and the dyeing cost is high. In the comparative example 1, only low concentration CHAPS (mass / volume concentration of 0.1%-1%) is used as a penetrating agent, and the dyeing agent has limited dyeing depth on the intact organ, only 373 μm. Further, using high concentration CHAPS (mass / volume concentration of 10%, 20%) as a penetrating agent can improve the dyeing depth of the intact organ, but the improvement effect is limited (dyeing depth of 481 μm), and when the concentration increases from 10% to 20% (concentration doubled), the dyeing depth only increases by 1.25 times (about 25%). It can be seen that the increase of dyeing depth is not proportional to the increase of concentration, and the doubling of concentration does not bring the same doubling of dyeing depth, but a small increase, which shows that the dyeing process has the characteristics of diminishing marginal effect, that is, with the further increase of the concentration of the dyeing agent, the improvement of the dyeing depth will gradually weaken. Therefore, the effect of simply increasing the amount of CHAPS to improve the dyeing depth is limited. In addition, CHAPS is expensive, which significantly increases the cost of cell construction dyeing of the intact organ. In the comparative example 2, only different concentrations of urea are used as penetrating agents, and when the mass / volume concentration of urea increases (0.1%-1%), the dyeing depth shows an upward trend; when it continues to increase to 10%, the dyeing depth decreases; when the concentration is greater than 10%, the dyeing depth shows an upward trend, and the maximum dyeing depth is 430 μm. In the comparative example 3, only different concentrations of 2,6-dimethyl-β-cyclodextrin are used as penetrating agents, and the dyeing agent has limited dyeing depth on the intact organ, and the dyeing process also has the characteristics of diminishing marginal effect.

[0052] Comparing the dyeing depth of Comparative Example 4 (total concentration of penetrating agent is 0.3%) and Comparative Example 1B (concentration of CHAPS is 0.3%), it can be seen from Table 1 and Table 4 that the dyeing effect of Comparative Example 4 is worse than that of Comparative Example 1B when the amount of penetrating agent is the same. Comparing the dyeing depth of Comparative Example 5 (total concentration of penetrating agent is 1.2%) and Comparative Example 2B (concentration of urea is 0.1%), it can be seen that the dyeing effect of Comparative Example 5 is only equal to that of Comparative Example 2B when the amount of penetrating agent is significantly increased. Comparing the dyeing depth of Comparative Example 6 (total concentration of penetrating agent is 10.2%) and Comparative Example 3E (concentration of 2,6-dimethyl-β-cyclodextrin is 10%), it can be seen that the dyeing effect of Comparative Example 6 is worse than that of Comparative Example 3E when the amount of penetrating agent is basically the same. Comparing the dyeing depth of Comparative Example 7 (total concentration of penetrating agent is 1.2%), Comparative Example 8 (total concentration of penetrating agent is 2.1%) and Comparative Example 1C (concentration of CHAPS is 1%), it can be seen that the dyeing effect of Comparative Examples 7 and 8 is still worse than that of Comparative Example 1C when the amount of penetrating agent is increased.

[0053] Comparing the dyeing depth of Examples 1-2 (total concentration of penetrating agent is 11.1%, and the concentration of CHAPS is 0.1%) and Comparative Example 1E (concentration of CHAPS is 10%), it can be seen that the dyeing effect of Examples 1-2 is basically equal to that of Comparative Example 1E, or even better than that of Comparative Example 1E (concentration of CHAPS is 10%) when the dyeing cost is significantly reduced.

[0054] Comparing the dyeing depth of Example 3 (total concentration of penetrating agent is 20.1%) and Comparative Example 1F (concentration of CHAPS is 20%), Comparative Example 2F (concentration of urea is 20%) and Comparative Example 3F (concentration of 2,6-dimethyl-β-cyclodextrin is 20%), it can be seen that the dyeing depth of Example 3 is 1.1 times that of Comparative Example 1F (single CHAPS), 1.3 times that of Comparative Example 2F (single urea component), and 1.4 times that of Comparative Example 3F (single 2,6-dimethyl-β-cyclodextrin) when the total concentration of penetrating agent is basically the same, which proves that the compounding of penetrating agents breaks the limit of single penetrating agent component to improve the penetration effect of dyeing agent, realizes the penetration effect that cannot be achieved by single penetrating agent component, and confirms the synergistic effect between the penetrating agent components.

[0055] Comparing the dyeing depth of Comparative Example 4 (total concentration of the penetration enhancer is 2.1%, concentration of CHAPS is 1%) with that of Comparative Example 1D (concentration of CHAPS is 3%), 2D (concentration of urea is 3%) and 3D (concentration of 2,6-dimethyl-β-cyclodextrin is 3%), it can be seen that, in the case of reducing the cost of dyeing and the amount of the penetration enhancer, the dyeing effect of Example 4 is 1.1 times that of Comparative Example 1D, 1.3 times that of Comparative Example 2D and 1.5 times that of Comparative Example 3D, achieving the penetration effect that cannot be achieved by a single penetration enhancer component and also confirming the synergistic effect between the penetration enhancer components.

[0056] Comparing the dyeing depth of Comparative Example 5 (total concentration of the penetration enhancer is 3%) with that of Comparative Example 1D (concentration of CHAPS is 3%), 2D (concentration of urea is 3%) and 3D (concentration of 2,6-dimethyl-β-cyclodextrin is 3%), it can be seen that, in the case of the same total concentration of the penetration enhancer, the dyeing depth of Example 5 is 1.2 times that of Comparative Example 1D, 1.4 times that of Comparative Example 2D and 1.6 times that of Comparative Example 3D, also confirming the synergistic effect between the penetration enhancer components.

[0057] Comparing the dyeing depth of Example 6 (total concentration of the penetration enhancer is 12%, concentration of CHAPS is 1%), Example 7 (total concentration of the penetration enhancer is 11.1%), Example 8 (total concentration of the penetration enhancer is 12%) and Example 9 (total concentration of the penetration enhancer is 21%) with that of Comparative Example 1D (concentration of CHAPS is 3%), it can be seen that, in the case of reducing the cost of dyeing, the dyeing depth of Examples 6-9 is better than that of Comparative Example 1D, and the dyeing effect of Examples 6 and 8 is even better than that of Comparative Example 1F (concentration of CHAPS is 20%), also confirming the synergistic effect between the penetration enhancer components.

[0058] Comparing the dyeing depth of Examples 6-8 with that of Comparative Example 2F (concentration of urea is 20%) and Comparative Example 3F (concentration of 2,6-dimethyl-β-cyclodextrin is 20%), it can be seen that, in the case of significantly reducing the amount of the penetration enhancer, the dyeing depth of Examples 6-8 is better than that of Comparative Example 2F and Comparative Example 3F, also confirming the synergistic effect between the penetration enhancer components.

[0059] Comparing the dyeing depth of Example 9 with that of Comparative Example 2G (concentration of urea is 30%) and Comparative Example 3G (concentration of 2,6-dimethyl-β-cyclodextrin is 30%), it can be seen that, in the case of significantly reducing the amount of the penetration enhancer, the dyeing depth of Example 9 is 1.1 times that of Comparative Example 2G and 1.4 times that of Comparative Example 3G, also confirming the synergistic effect between the penetration enhancer components.

[0060] Comparing the dyeing depth of Example 10 (CHAPS concentration in the penetration enhancer is 10%, total concentration of the penetration enhancer is 11.1%), Example 11 (total concentration of the penetration enhancer is 20.1%), Example 12 (total concentration of the penetration enhancer is 11.1%), Example 13 (total concentration of the penetration enhancer is 12%), Example 14 (total concentration of the penetration enhancer is 21%), Example 15 (total concentration of the penetration enhancer is 20.1%), Example 16 (total concentration of the penetration enhancer is 21%) and Example 17 (total concentration of the penetration enhancer is 30%) with Comparative Example 1F (CHAPS concentration is 20%), it can be seen that, under the condition of significantly reducing the dyeing cost, the dyeing depth of Examples 10-15 reaches 1.1 times, 1.2 times, 1.2 times, 1.3 times, 1.5 times, 1.4 times of Comparative Example 1F respectively, which also confirms the synergistic effect among the components of the penetration enhancer, and the dyeing depth of Examples 16-17 even reaches 2 times, 2 times of Comparative Example 1F.

[0061] Comparing the dyeing depth of Examples 10-17 with Comparative Example 2G (urea concentration is 30%), it can be seen that, under the condition of significantly reducing the amount of the penetration enhancer, the dyeing depth of Examples 10-17 reaches 1.3 times, 1.4 times, 1.3 times, 1.4 times, 1.6 times, 1.6 times of Comparative Example 2G respectively, which also confirms the synergistic effect among the components of the penetration enhancer, and the dyeing depth of Examples 16-17 even reaches 2.2 times, 2.3 times of Comparative Example 2G.

[0062] Comparing the dyeing depth of Examples 10-17 with Comparative Example 3G (2,6-dimethyl-β-cyclodextrin concentration is 30%), it can be seen that, under the condition of significantly reducing the amount of the penetration enhancer, the dyeing depth of Examples 10-17 reaches 1.6 times, 1.7 times, 1.6 times, 1.8 times, 2.0 times, 1.9 times, 2.7 times, 2.8 times of Comparative Example 3G respectively, which also confirms the synergistic effect among the components of the penetration enhancer.

[0063] It can be seen from Tables 1-4 that, when the concentration of the detergent in the penetration dyeing reagent is 0.1%-1% (w / v), the concentration of the super-hydrating agent and the cholesterol removing agent is independently 1%-20% (w / v) respectively and the total concentration of the two is greater than 2% (w / v); or when the concentration of the detergent CHAPS in the penetration dyeing reagent is 10%, the concentration of the super-hydrating agent and the cholesterol removing agent is 0.1%-20% (w / v) and the total concentration of the two is greater than 0.2% (w / v), the penetration of the dyeing agent into the complete organ can be significantly improved under the condition of reducing the dyeing cost and reducing the amount of the penetration enhancer, the limit of improving the penetration effect of the dyeing agent by a single penetration enhancer component is broken by compounding the three types of penetration enhancers, the penetration effect that cannot be achieved by a single penetration enhancer component is achieved, and the synergistic effect among the components of the penetration enhancer is confirmed.

[0064] In summary, the present application can significantly improve the dyeing depth of the dyeing agent on the intact organ by compounding the detergent (CHAPS), the super-hydrating agent (urea) and the cholesterol removing agent (2, 6-dimethyl-β-cyclodextrin) and regulating the appropriate amount of each component. The analysis reason may be that the detergent in the transmittance enhancing dyeing reagent has hydrophilic group and hydrophobic group at both ends of the molecule, which can self-assemble into micelles in water, wrap hydrophobic molecules inside, form micelles with cell membrane phospholipids and form holes on the membrane, effectively improve the cell membrane permeability; at the same time, the cyclodextrin substance as a cholesterol removing agent has a high affinity with the cholesterol in the cell membrane, can remove a large amount of membrane cholesterol, reduce the stability of the membrane structure, enhance the permeability, and more easily form micelles with the detergent, further improve the membrane permeability. In addition, the super-hydrating agent containing a large number of amino groups has a stronger hydrogen bonding ability than water, can competitively replace the water between macromolecular proteins, cause the disintegration of the high-order structure of collagen fibers, cell skeleton and other structures, and affect the arrangement of membrane phospholipids, thereby improving the cell membrane permeability. Through the synergistic effect of the components in the composite transmittance enhancer, the transmittance enhancing dyeing reagent prepared by mixing the above transmittance enhancing dyeing reagent with the dyeing agent can effectively improve the permeability of the macromolecular dyeing agent to the intact biological tissue when the cell structure is dyed, thereby significantly improving the dyeing speed of the intact biological tissue and effectively improving the dyeing uniformity.

[0065] Application Example The transmittance enhancing dyeing reagent used in the present application example is obtained by uniformly mixing a composite transmittance enhancer and a dyeing agent, wherein the concentration of the dyeing agent propidium iodide (PI) is 100 μg / mL; the concentration of the transmittance enhancer CHAPS is 10% (w / v), the concentration of urea is 20% (w / v), and the concentration of 2, 6-dimethyl-β-cyclodextrin is 1% (w / v); and the rest is water. Then, the above transmittance enhancing dyeing reagent is used to realize the cell structure labeling of the intact organ by passive soaking and to perform high-resolution three-dimensional imaging, which specifically includes the following steps: (1) Pretreatment One two-month-old C57 mouse is taken, and the target organ is specifically labeled by tail vein injection in advance. After the intravascular fluorescent marker is fully circulated and labeled, the mouse is anesthetized by intraperitoneal injection of a mixture of 10% urethane and 2% chloral hydrate. Then, the chest is opened to expose the heart, the right auricle is cut open, and a needle is inserted into the left ventricle from the apex of the heart. The 37℃ PBS solution is perfused at a speed of about 15 mL / min for 3-5 min. After the liver is observed to be whitened, the 4% PFA solution at 4℃ is perfused at the same speed for 5 min. After the mouse limbs and tail are observed to be rigid, it is indicated that the perfusion is sufficient. The intact target organ (mouse brain) is taken out and fixed in the 4% PFA solution at 4℃ for 12 h to obtain the pretreated intact organ.

[0066] (2) Cell structure dyeing The fixed intact mouse brain (the size of length, width and thickness is 15mmx10mmx6mm) is placed in the penetration dyeing reagent for dyeing at room temperature, the dyeing time is 5 days, after the dyeing is completed, the intact mouse brain is placed in the PBS solution for cleaning for 1h, and the cleaning is repeated for 3 times. The PI solution with a concentration of 100μg / mL is used for cell structure dyeing in the control group.

[0067] (3) Three-dimensional imaging The dyed intact mouse brain is embedded, and then fixed at a specified position of a fluorescence micro-optical sectioning tomography system, continuous collection is carried out, and the collected data is directly observed.

[0068] Figure 1 The maximum coronal plane cell structure image of the intact mouse brain treated by the penetration dyeing reagent provided by the embodiment and obtained by the fluorescence micro-optical sectioning tomography system, Figure 2 The maximum coronal plane cell structure image of the intact mouse brain treated by the PI solution of the control group and obtained by the fluorescence micro-optical sectioning tomography system can be seen, the penetration dyeing reagent provided by the application can quickly and high-quality complete the cell structure labeling of the intact biological tissue by the passive soaking method, the dyeing time of the intact organ is obviously shortened, and the dyeing uniformity is excellent.

[0069] In addition, compared with the existing penetration dyeing reagent, the penetration dyeing reagent provided by the application can be compatible with various cell structure dyes to realize different specific fluorescence dyeing schemes, can also be adapted to different sizes of intact biological tissues, realize the rapid dyeing of the intact biological tissue and avoid the damage of the biological tissue, has the advantages of simple operation, strong universality, low dyeing cost, short dyeing time, excellent dyeing effect and the like.

[0070] It is easy for those skilled in the art to understand that the above-mentioned is only the preferred embodiment of the application, and does not limit the application, any modification, equivalent replacement and improvement made within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. A penetration enhancing staining reagent suitable for rapid labeling of intact organ cell constructs, characterized in that, The composite penetration enhancer, the dyeing agent and the solvent are included. The composite penetration enhancer is compounded by a detergent, a super-hydrating agent and a cholesterol removing agent, and is used to improve the penetration of the dyeing agent into the intact organ.

2. The transmittance increasing dyeing reagent according to claim 1, characterized in that, The detergent is one or more of CHAPS, Triton X-100, Tween 20, saponin and SDS; and / or, The super-hydrating agent is one or more of urea, formamide, 1,3-cyclohexanedimethylamine and m-xylylenediamine; and / or, The cholesterol removing agent is one or more of β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, cholesterol oxidase, digitonin and phelliphen.

3. The transmittance increasing dyeing reagent according to claim 1, characterized by, When the concentration of the detergent in the penetration dyeing reagent is 0.1%-1%(w / v), the concentration of the super-hydrating agent and the cholesterol removing agent is independently 1%-20%(w / v), and the total concentration of the super-hydrating agent and the cholesterol removing agent is greater than 2%(w / v).

4. The transmittance increasing dyeing reagent according to claim 1, characterized by, When the concentration of the detergent in the penetration dyeing reagent is greater than 1%(w / v), the concentration of the super-hydrating agent and the cholesterol removing agent is independently 0.1%-20%(w / v), and the total concentration of the super-hydrating agent and the cholesterol removing agent is greater than 0.2%(w / v).

5. The anti-reflective dyeing reagent according to claim 4, characterized in that, The concentration of the detergent in the penetration dyeing reagent is greater than 1%(w / v) and less than 20%(w / v).

6. The transmittance increasing dyeing reagent according to claim 1, wherein The dyeing agent is one or more of a fluorescent nuclear marker, a protein marker, fluorescent Nissl dye and a lectin fluorescent conjugate; and / or, The concentration of the dyeing agent in the penetration dyeing reagent is 1 μg / mL-200 μg / mL; and / or, The solvent is deionized water, phosphate buffer, cell culture buffer or a combination thereof.

7. A method of rapid labeling of a transparentized organ suitable for whole organ cell construction, characterized by, The method comprises the following steps: The pre-processed intact organ is soaked in the penetration dyeing reagent as claimed in any one of claims 1-6 to achieve cell architecture labeling of the intact organ.

8. The method of claim 7, wherein, The pre-processing method comprises immunostaining, chemical dye staining, virus marker staining or transgenic staining of the intact organ.

9. The anti-reflective coating method according to claim 7, characterized in that, The penetration method further comprises three-dimensional imaging of the intact organ with cell architecture labeling, and the three-dimensional imaging comprises the following steps: The intact organ with cell architecture labeling is embedded, and then the embedded intact organ is imaged; The imaging method comprises fluorescent microscopic optical section tomography, optical transparent tissue sheet imaging or two-photon sequential tomography.

10. Use of the penetration dyeing reagent as claimed in any one of claims 1-6 in three-dimensional reconstruction spatial positioning, cell counting and morphological study in physiological and pathological research, and precise anatomical atlas drawing.

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