Biological tissue transparentizing kit, imaging method and application

Abstract

The invention belongs to the technical field of biology, and discloses a biological tissue transparentizing kit, an imaging method and application, the biological tissue transparentizing kit comprises a biological tissue transparentizing agent and an antibody incubation agent, and the antibody incubation agent comprises a first incubation agent and a second incubation agent; according to the tissue transparentizing reagent disclosed by the invention, the permeability of antibodies in tissues is optimized, the antibodies are uniformly distributed in the tissues, and the efficiency and effect of tissue transparentizing treatment and subsequent immunolabeling are greatly improved; the tissue transparentizing imaging method can enhance the tissue transparentizing efficiency and degree, is especially suitable for integrated visual detection of multiple tissues such as blood vessels, nerves, lymphatic vessels, muscles, osteochondral tissues and the like of a motor system, and has a better detection effect on proteins with low expression quantity and unstable properties.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a biological tissue transparentization kit and an imaging method and application. Background Art

[0002] Traditional histological techniques usually stain tissue sections to depict the microstructure in the tissue, but due to the limitations of its analytical field of view, the overall information of the sample will be lost. Continuous sectioning of the tissue or preparation of thick tissue sections, and then reconstruction and construction of a three-dimensional image of tissue proteins using a laser confocal microscope is not only time-consuming and labor-intensive, but also inevitably results in the loss of structural information, and is only applicable to smaller tissues. The development and application of advanced imaging technologies such as computed tomography, magnetic resonance imaging, positron emission tomography, and near-infrared imaging have revealed the anatomical structure of living animals, but due to the lack of high-contrast cell labeling tools, these imaging technologies cannot achieve single-cell or even molecular level characteristic analysis. Although two-photon fluorescence microscopy and confocal microscopy can increase the depth of tissue imaging to hundreds of microns, the light absorption and light scattering of biological tissues still limit the application conditions of these technologies.

[0003] Tissue clearing technology has developed rapidly in the past few years. Many advanced tissue clearing technologies have emerged and are widely used in various fields, especially in diseases related to the nervous system. However, the tissue heterogeneity of the musculoskeletal system is high. In addition to the vascular system such as nerves, blood vessels, lymphatic vessels, and soft tissues such as muscles, ligaments, and tendons, it also includes bones and cartilage hard tissues. Compared with soft tissues, bone tissue is highly mineralized, rich in collagen, has a relatively low lipid content, and contains highly colored blood cells in the bone marrow space. These characteristics limit the application of many tissue optical clearing methods to bone tissue. In addition, since the rich extracellular matrix such as collagen, proteoglycans, and hyaluronic acid in bone and joint tissues will form a dense barrier, which seriously affects the penetration of antibodies into the tissue, the current clearing-related technologies can only achieve immunolabeling and imaging of proteins in the superficial parts of bone and joint tissues (such as neurovascular proteins in the periosteum). To achieve overall imaging of proteins inside bone and joint tissues, fluorescent proteins are currently mostly used to label animal tissues. To avoid the disadvantage of insufficient antibody penetration during antibody labeling, fluorescent proteins are directly integrated into the animal body through genetic engineering technology, so that the target tissue or cells can emit fluorescence under the excitation of light of a specific wavelength, which is convenient for observation and tracking. However, the cost and time cost of constructing transgenic animals are too high; and recent studies have used a large amount of collagenase to pre-treat bone tissue to improve the permeability of antibodies, but this method will destroy the integrity of the tissue structure and the application scenarios are limited (such as the inability to label and detect soft tissue structures such as periosteum and synovium and extracellular matrix). Therefore, how to improve the efficiency of overall tissue transparency and antibody labeling to achieve integrated and visualized imaging of tissue structures such as blood vessels, nerves, muscles, and bones in the locomotor system is a technical bottleneck that urgently needs to be broken through. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a biological tissue transparentization kit and an imaging method and application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a biological tissue clearing kit, comprising a biological tissue permeabilizing agent and an antibody incubating agent; the antibody incubating agent comprises a first incubating agent and a second incubating agent; the biological tissue permeabilizing agent or the second incubating agent comprises 2,6-di-O-methyl-β-cyclodextrin; the first incubating agent comprises 2,6-di-O-methyl-β-cyclodextrin, N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine and urea.

[0007] The biological tissue clearing kit of the present invention not only optimizes the permeability and uniform distribution of antibodies in tissues through the mutual coordination of reagents containing multiple components, but also cleverly regulates the binding force between antigens and antibodies, greatly improving the efficiency and effect of tissue clearing treatment and subsequent immunolabeling. By adding 2,6-di-O-methyl-β-cyclodextrin to the biological tissue permeabilizer to remove cholesterol from the tissue and enhance the permeability of antibodies, 2,6-di-O-methyl-β-cyclodextrin is added to the first incubator and the second incubator to reduce the aggregation of antibodies during the subsequent antibody incubation process, thereby achieving uniform distribution and effective utilization of antibodies inside and outside the tissue, and at the same time, urea and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine are added to the first incubator and coordinated with other components to weaken the affinity of antigens and antibodies during the initial antibody incubation process, thereby reducing The local binding of antigen and antibody is reduced, thereby avoiding the waste and local consumption of antibodies. Subsequently, the second incubation agent without urea and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine is added to restore the affinity of antigen and antibody in the subsequent antibody incubation stage, so as to realize the conversion process of antigen-antibody binding "off"-"on" to reduce the local consumption and local uneven aggregation of antibodies, thereby enhancing the freeness and permeability of antibodies, realizing the uniform distribution and effective utilization of antibodies inside and outside the tissue, thereby improving the efficiency of overall tissue transparency and antibody labeling.

[0008] As a preferred embodiment of the biological tissue clearing kit of the present invention, the biological tissue clearing kit also includes a cleaning agent and a refractive index matching agent; the cleaning agent includes a non-ionic surfactant with a volume percentage of 0.01%-0.2% and a heparin solution with a concentration of 5μg / mL-20μg / mL; preferably, the non-ionic surfactant is TritonX-100; the refractive index matching agent includes an antioxidant with a volume percentage of 0.1%-1%; preferably, the antioxidant is vitamin E.

[0009] The nonionic surfactant in the cleaning agent of the biological tissue transparentization kit of the present invention is used to reduce background signals and enhance the specific binding of antibodies to target antigens; the heparin solution is used to enhance the permeability of antibodies to tissues, making it easier for antibodies to penetrate tissue barriers and reach deeper tissue layers; and the antioxidant in the refractive index matching agent is used to improve the stability of fluorescent signals.

[0010] Preferably, the cleaning agent further comprises 2.5%-20% by volume of glycerol.

[0011] Glycerol is added to the cleaning agent of the biological tissue clearing kit of the present invention to prepare another cleaning agent, and the two cleaning agents are used alternately to clean the tissue in the antibody cleaning process of the biological tissue clearing imaging method. The osmotic pressure difference formed by the glycerol content is used to enhance the fluidity of the liquid and improve the antibody cleaning efficiency, and glycerol is used to reduce the aggregation of free antibodies.

[0012] As a preferred embodiment of the biological tissue clearing kit described in the present invention, the biological tissue permeabilization agent includes 0.1%-1% 2,6-di-O-methyl-β-cyclodextrin by mass volume; the first incubation agent includes 0.1%-1% 2,6-di-O-methyl-β-cyclodextrin by mass volume, 2.5%-5% N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine by volume, and 0.5mol / L-2mol / L urea; the second incubation agent includes 0.1%-1% 2,6-di-O-methyl-β-cyclodextrin by mass volume.

[0013] Preferably, the biological tissue permeabilizing agent includes 1% by mass volume of 2,6-di-O-methyl-β-cyclodextrin; the first incubation agent includes 1% by mass volume of 2,6-di-O-methyl-β-cyclodextrin, 2.5% by volume of N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, and 1 mol / L urea; the second incubation agent includes 1% by mass volume of 2,6-di-O-methyl-β-cyclodextrin.

[0014] As a preferred embodiment of the biological tissue clearing kit of the present invention, the biological tissue permeabilization agent also includes a non-ionic detergent with a volume percentage of 0.1%-15%, an ionic detergent with a mass volume percentage of 0.1%-1%, an amino acid with a concentration of 10mmol / L-50mmol / L, dimethyl sulfoxide with a volume percentage of 1%-10%, ethylenediaminetetraacetic acid with a concentration of 10mmol / L-50mmol / L, and a phosphatase inhibitor with a concentration of 0.1mmol / L-1mmol / L; preferably, the non-ionic detergent is Triton X-100, the ionic detergent is sodium deoxycholate, and the amino acid is glycine.

[0015] The nonionic detergent and the ionic detergent in the biological tissue permeabilizing agent of the biological tissue transparentizing kit of the present invention are used to destroy the double-layer phospholipid membrane of tissue cells, thereby releasing and dissolving membrane-bound proteins; amino acids are used to reduce background non-specific staining and enhance fluorescent signals; dimethyl sulfoxide is used to enhance biological membrane permeability; ethylenediaminetetraacetic acid is used as an inhibitor of nuclease and protease; and phosphatase inhibitors protect the stability of phosphorylated proteins.

[0016] As a preferred embodiment of the biological tissue clearing kit of the present invention, the first incubation agent or the second incubation agent also includes 2.5%-20% glycerol by volume; preferably, the first incubation agent also includes 20% glycerol by volume; preferably, the second incubation agent also includes 10% glycerol by volume.

[0017] The glycerol in the first incubation solution and the second incubation solution of the biological tissue clearing kit of the present invention is used to cooperate with 2,6-di-O-methyl-β-cyclodextrin to reduce the aggregation of antibodies in the subsequent antibody incubation process, thereby achieving uniform distribution and effective utilization of antibodies inside and outside the tissue.

[0018] As a preferred embodiment of the biological tissue clearing kit of the present invention, the first incubator also includes a non-ionic surfactant with a volume percentage of 0.1%-10%, a heparin solution with a concentration of 5μg / mL-20μg / mL, an amino acid with a concentration of 10mmol / L-50mmol / L, a salt with a mass volume percentage of 0.2%-0.5%, and a blocking agent with a volume percentage of 1%-5%; preferably, the non-ionic surfactant is Triton X-100, the amino acid is glycine, the salt is sodium chloride, and the blocking agent is donkey serum; the second incubator includes a non-ionic surfactant with a volume percentage of 0.1%-0.5%, a heparin solution with a concentration of 5μg / mL-20μg / mL, an amino acid with a concentration of 10mmol / L-50mmol / L, and a salt with a mass volume percentage of 0.2%-0.5%; preferably, the non-ionic surfactant is Triton X-100, the amino acid is glycine, and the salt is sodium chloride.

[0019] In the first incubation solution of the biological tissue transparent kit of the present invention, the nonionic surfactant is used to reduce the background signal and enhance the specific binding of the antibody and the target antigen; the heparin solution is used to enhance the permeability of the antibody to the tissue, so that the antibody can more easily penetrate the tissue barrier and reach a deeper tissue layer; the salt is used to enhance the ionic strength of the solution, reduce the antigen-antibody affinity, and thus reduce the local uneven aggregation of the antibody; the amino acid can be used to reduce the background nonspecific staining and enhance the fluorescence signal; the blocking agent is used to nonspecifically bind to the protein, thereby reducing the background signal and reducing the occurrence of false positive results; and urea and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine are not added to the second incubation solution, and the antigen-antibody affinity is restored in the subsequent antibody incubation stage to realize the antigen-antibody binding "off"-"on" conversion process to reduce the local consumption and local uneven aggregation of the antibody, thereby enhancing the antibody's freeness and permeability, and realizing the uniform distribution and effective utilization of the antibody inside and outside the tissue, thereby improving the efficiency of overall tissue transparentization and antibody labeling.

[0020] In a second aspect, the present invention provides an application of the biological tissue clearing kit in biological tissue clearing imaging; preferably, the biological tissue is any one of vascular tissue, neural tissue, lymphatic tissue, muscle tissue, and osteochondral tissue of the musculoskeletal system.

[0021] The biological tissue clearing kit of the present invention can be specifically applied to clearing imaging of in vitro biological tissues, and has a good detection effect on proteins with low expression levels and instability. The in vitro biological tissue is any one of musculoskeletal system tissues (such as bones, joints, and spine), central and peripheral nervous tissues (such as brain and spinal cord), digestive system tissues (such as liver and intestines), circulatory system tissues (such as heart and kidneys), urinary or reproductive system tissues (such as kidney and ovarian tissues), and embryos. The biological tissue clearing kit of the present invention is used for biological tissue clearing treatment or biological tissue clearing imaging through its unique combination of components and concentration ratios, and can achieve clearing of a variety of organisms and a variety of tissues, and is particularly suitable for integrated visualization detection of multiple tissues such as blood vessels, nerves, lymphatic vessels, muscles, and bone and cartilage tissues of the musculoskeletal system.

[0022] In a third aspect, the present invention provides a method for transparentizing and imaging biological tissues, comprising the following steps: (i) cleaning and fixing biological tissues; performing antigen repair; and then dehydrating, degreasing, and decolorizing the tissues; (ii) using the biological tissue permeabilizing agent to permeabilize the tissues, dehydrating and drying to obtain dried tissues; (iii) using the first incubating agent to dilute the antibodies to obtain a preliminary antibody incubation solution, incubating the tissues for 2h-24h, adding the second incubating agent, and continuing to incubate for 6h-24h; cleaning, dehydrating, and imaging.

[0023] The biological tissue transparent imaging method of the present invention advances the defatting step to before decolorization, which can avoid the influence of excess lipid on antibody diffusion, and uses the biological tissue transparent kit of the present invention to transparentize the tissue, thereby improving the permeability of the antibody, thereby improving the efficiency of tissue immunolabeling and transparentization, and can be used for integrated visualization detection of multiple tissues such as blood vessels, nerves, lymphatic vessels, muscles, and osteochondral tissues; and when the tissue is a tissue containing bone structure (except brain tissue, liver, tumor, etc.), decalcification can be performed before the antigen repair described in step (i); if the tissue is a transgenic animal tissue of a spontaneous fluorescent protein or an animal tissue transfected with a fluorescent protein using a virus as a vector, the antibody incubation process of step (iii) is not performed.

[0024] As a preferred embodiment of the biological tissue clearing imaging method of the present invention, in step (iii), the first incubation agent is used to dilute the primary antibody to obtain a primary antibody preliminary incubation solution, the tissue is incubated for 2h-24h, the second incubation agent is added, and the incubation is continued for 6h-24h; washing, dehydration and drying; the first incubation agent is used to dilute the secondary antibody to obtain a secondary antibody preliminary incubation solution, the tissue is incubated for 2h-24h, the second incubation agent is added, and the incubation is continued for 6h-24h; washing, dehydration, and imaging; further preferably, the volume ratio of the first incubation agent to the primary antibody is (10-50):1; the volume ratio of the second incubation agent to the secondary antibody is (20-100):1.

[0025] In the biological tissue transparentization imaging method of the present invention, the antibody is any one of a protein, a nanobody, a monoclonal antibody, a polyclonal antibody and an in situ fluorescent RNA probe coupled to a fluorophore, and when the primary antibody can be directly used to detect the target antigen, the secondary antibody incubation in step (iv) is not performed.

[0026] As a preferred embodiment of the biological tissue clearing imaging method of the present invention, in the steps (iii) and / or (iv), the cleaning is performed by alternating treatment with the two cleaning agents.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: first, the biological tissue clearing kit of the present invention can effectively improve the permeability of antibodies, shorten the processing time of tissues, greatly improve the efficiency of tissue immunolabeling and clearing, and is suitable for bones and joint tissues that are difficult to label and clear imaging; it effectively reduces antibody aggregation and local consumption phenomena; secondly, the biological tissue clearing imaging method of the present invention is compatible with virus tracing, fluorescent dyes, fluorescently labeled animals and in situ hybridization, has a good detection effect on proteins with low expression levels and unstable properties, and the materials are easily available, the process is simple, and the applicability is wide. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the chemical structure of the components in the biological tissue clearing kit of the present invention: (A) 2,6-di-O-methyl-β-cyclodextrin, (B) N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, (C) urea, (D) dibenzyl ether;

[0029] Figure 2 is an operation flow chart of the biological tissue clearing imaging method of the present invention;

[0030] Figure 3 This is a general picture of the vascular tissue of the whole knee joint of a mouse after being processed by the biological tissue clearing imaging method in Experimental Example 1 of the present invention;

[0031] Figure 4Schematic diagram of the biological tissue clearing kit prepared according to Examples 2-4 of the present invention: a is Example 2; b is Example 3; c is Example 4;

[0032] Figure 5 This is the imaging effect diagram of tissue transparency of mouse knee joint vascular tissue after Anti-CD31 immunolabeling in Experimental Example 1 of the present invention: (left) three-dimensional imaging effect diagram, (right) representative section imaging diagram;

[0033] Figure 6 This is the imaging effect diagram of the tissue transparency of the whole knee joint vascular tissue of mice after Anti-CGRP immunolabeling in Experimental Example 2 of the present invention: (left) three-dimensional imaging effect diagram, (right) representative section imaging diagram;

[0034] Figure 7 This is a comparison chart of the percentage of biological tissue transparency marking in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0035] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, equipment, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0037] In the description of this application, it should be noted that the term "volume percentage" refers to the percentage of the volume of a substance in the total volume of a solution, "mass volume percentage" refers to the percentage of the mass (g) of a substance in the total volume (mL) of a solution, and "volume ratio" refers to the volume ratio of two solutions.

[0038] The present application provides a biological tissue transparent imaging method, comprising the following steps:

[0039] (i) Cleaning and fixing biological tissues; decalcifying and performing antigen repair; and then dehydrating, degreasing and decolorizing the tissues;

[0040] (ii) using the biological tissue permeabilizing agent to permeabilize the tissue, dehydrating and drying to obtain a dried tissue;

[0041] (iii) using the first incubation agent to dilute the antibody to obtain a preliminary antibody incubation solution, incubating the tissue for 2h-24h, adding the second incubation agent, and continuing to incubate for 6h-24h; washing, dehydrating, and imaging.

[0042] In one embodiment, the biological tissue is at least one of a reptile, an avian, a mammalian organism or a tissue engineering biomaterial.

[0043] In one embodiment, in step (i), the cleaning is first performed by perfusion solution and then by flushing solution; the perfusion solution is a heparin solution, the total amount of the heparin solution is 50UI-100UI, the flushing solution is a PBS solution, the volume of the PBS solution is 15mL-150mL.

[0044] In one embodiment, in step (i), the flushing solution and the fixative solution are frozen to 4° C. before perfusion.

[0045] In one embodiment, in step (i), the fixing solution used for fixation is at least one of paraformaldehyde, glutaraldehyde, and formalin.

[0046] In one embodiment, in step (i), the fixing solution used for the fixation is a paraformaldehyde solution, the volume of the paraformaldehyde solution is 15 mL-150 mL, and the mass volume percentage is 1%-4%.

[0047] In one embodiment, in step (i), the fixing time is 4 h-12 h, and the fixing temperature is room temperature or 4°C.

[0048] In one embodiment, in step (i), the decalcification solution used for decalcification is at least one of a formic acid-formalin solution, ethylenediaminetetraacetic acid, Von Ebnei solution, and a nitric acid-containing decalcification solution.

[0049] In one embodiment, in step (i), the decalcification solution used for decalcification is an ethylenediaminetetraacetic acid solution, and the concentration of the ethylenediaminetetraacetic acid solution is 350mmol / L-500mmol / L.

[0050] In one embodiment, in step (i), the decalcification time is 3 days to 28 days, and the decalcification temperature is room temperature or 37°C.

[0051] In one embodiment, in step (i), the antigen retrieval temperature is 65°C-100°C, and the time is 10 min-30 min.

[0052] In one embodiment, in step (i), the degreasing solution used for degreasing comprises dichloromethane and methanol; the volume ratio of dichloromethane to methanol is (1-2):1, and the degreasing time is 12h-48h.

[0053] In one embodiment, in step (i), the decolorization solution used for decolorization includes hydrogen peroxide (30% by volume) and methanol, the volume ratio of hydrogen peroxide to methanol is 1:(5-10), and the decolorization time is 8h-24h.

[0054] In one embodiment, in step (ii), the pH value of the biological tissue permeabilization agent is 6.5-7.4.

[0055] In one embodiment, in step (ii), the permeabilization time is 8h-24h, and the permeabilization is performed by replacing fresh reagent A every 8h-12h.

[0056] In one embodiment, in step (ii), the evaporation drying time is 15 min-60 min.

[0057] In one embodiment, in step (iii), the pH value of the first incubation agent is 6.5-7.4.

[0058] In one embodiment, in step (iii), the pH value of the second incubation agent is 6.5-7.4.

[0059] In one embodiment, in step (iii), the pH value of the cleaning agent is 6.5-7.4.

[0060] In one embodiment, in step (iii), the interval between the alternating use of two cleaning agents for cleaning is 4h-12h, and the total cleaning time is 24h-72h.

[0061] In one embodiment, in step (iii), the refractive index matching agent is added before imaging to perform refractive index matching.

[0062] In one embodiment, in step (iii), the refractive index matching time is 12h-72h, and the refractive index matching agent is replaced with a fresh one every 6 hours.

[0063] In one embodiment, in step (iii), the microscope used for imaging is any one of a wide-field fluorescence microscope, a laser sheet scanning microscope, a laser confocal microscope and a two-photon fluorescence microscope.

[0064] Example 1: Preparation of tissue clearing kit

[0065] (1) Reagent A: Using PBS solution as solvent, prepare a biological tissue permeabilizer consisting of 10% by volume Triton X-100, 0.1% by volume sodium deoxycholate, 10% by volume dimethyl sulfoxide, 25 mmol / L ethylenediaminetetraacetic acid, 1% by volume 2,6-di-O-methyl-β-cyclodextrin, 0.1 mmol / L phosphatase inhibitor, and 50 mmol / L glycine, and adjust the pH value to 7.2.

[0066] (2) Reagent B: Using PBS solution as solvent, prepare a primary antibody incubation agent containing 5% by volume of Triton X-100, 0.35% by volume of sodium chloride, 10 μg / mL of heparin solution, 3% by volume of donkey serum, 20% by volume of glycerol, 1% by volume of 2,6-di-O-methyl-β-cyclodextrin, 2.5% by volume of N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, 1 mol / L of urea, and 15 mmol / L of glycine, and adjust the pH value to 7.2.

[0067] (3) Reagent C: Using PBS solution as solvent, prepare a second antibody incubation agent containing 0.2% by volume of Triton X-100, 10 μg / mL of heparin solution, 0.35% by volume of sodium chloride, 10% by volume of glycerol, 1% by volume of 2,6-di-O-methyl-β-cyclodextrin, and 15 mmol / L of glycine, and adjust the pH value to 7.2.

[0068] (4) Reagent D: Using PBS solution as solvent, prepare a cleaning solution containing 0.1% by volume of Triton X-100 and 10 μg / mL of heparin solution, and add 20% by volume of glycerol to reagent D to prepare reagent D2.

[0069] (5) Reagent E: Using benzyl ether as solvent, prepare a refractive index matching agent containing 0.5% by volume of vitamin E.

[0070] Example 2: Preparation of tissue clearing kit

[0071] (1) Reagent A: Using PBS solution as solvent, prepare a biological tissue permeabilizer consisting of 10% by volume Triton X-100, 0.1% by volume sodium deoxycholate, 10% by volume dimethyl sulfoxide, 25 mmol / L ethylenediaminetetraacetic acid, 0.5% by volume 2,6-di-O-methyl-β-cyclodextrin, 0.1 mmol / L phosphatase inhibitor, and 50 mmol / L glycine, and adjust the pH value to 7.2.

[0072] (2) Reagent B: Using PBS solution as solvent, prepare a primary antibody incubation agent containing 5% by volume of Triton X-100, 0.35% by volume of sodium chloride, 10 μg / mL of heparin solution, 3% by volume of donkey serum, 10% by volume of glycerol, 0.5% by volume of 2,6-di-O-methyl-β-cyclodextrin, 4% by volume of N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, 1 mol / L of urea, and 15 mmol / L of glycine, and adjust the pH value to 7.2.

[0073] (3) Reagent C: Using PBS solution as solvent, prepare a secondary antibody incubation agent containing 0.2% by volume of Triton X-100, 10 μg / mL heparin solution, 0.35% by volume of sodium chloride, 5% by volume of glycerol, 0.5% by volume of 2,6-di-O-methyl-β-cyclodextrin, and 15 mmol / L of glycine, and adjust the pH value to 7.2.

[0074] (4) Reagent D: Using PBS solution as solvent, prepare a cleaning solution containing 0.1% by volume of Triton X-100 and 10 μg / mL of heparin solution, and add 20% by volume of glycerol to reagent D to prepare reagent D2.

[0075] (5) Reagent E: Using benzyl ether as solvent, prepare a refractive index matching agent containing 0.5% by volume of vitamin E.

[0076] Example 3: Preparation of tissue clearing kit

[0077] (1) Reagent A: Using PBS solution as solvent, prepare a biological tissue permeabilizer consisting of 1% by volume Triton X-100, 0.1% by volume sodium deoxycholate, 1% by volume dimethyl sulfoxide, 10 mmol / L ethylenediaminetetraacetic acid, 0.1% by volume 2,6-di-O-methyl-β-cyclodextrin, 0.1 mmol / L phosphatase inhibitor, and 10 mmol / L glycine, and adjust the pH value to 7.2.

[0078] (2) Reagent B: Using PBS solution as solvent, prepare a first incubation agent containing 0.5% by volume Triton X-100, 0.2% by volume sodium chloride, 5 μg / mL heparin solution, 1% by volume donkey serum, 5% by volume glycerol, 0.1% by volume 2,6-di-O-methyl-β-cyclodextrin, 2.5% by volume N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, 0.5 mol / L urea, and 10 mmol / L glycine, and adjust the pH value to 7.2.

[0079] (3) Reagent C: Using PBS solution as solvent, prepare a second incubation agent containing 0.1% by volume of Triton X-100, 5 μg / mL heparin solution, 0.2% by volume of sodium chloride, 2.5% by volume of glycerol, 0.1% by volume of 2,6-di-O-methyl-β-cyclodextrin, and 10 mmol / L of glycine, and adjust the pH value to 7.2.

[0080] (4) Reagent D: Using PBS solution as solvent, prepare a cleaning solution containing 0.01% by volume of Triton X-100 and 10 μg / mL of heparin solution, and add 5% by volume of glycerol to reagent D to prepare reagent D2.

[0081] (5) Reagent E: Using benzyl ether as solvent, prepare a refractive index matching agent containing 0.1% by volume of vitamin E.

[0082] Example 4: Preparation of tissue clearing kit

[0083] (1) Reagent A: Using PBS solution as solvent, prepare a biological tissue permeabilizer consisting of 15% by volume Triton X-100, 1% by volume sodium deoxycholate, 10% by volume dimethyl sulfoxide, 50 mmol / L ethylenediaminetetraacetic acid, 1% by volume 2,6-di-O-methyl-β-cyclodextrin, 1 mmol / L phosphatase inhibitor, and 50 mmol / L glycine, and adjust the pH value to 7.2.

[0084] (2) Reagent B: Using PBS solution as solvent, prepare a first incubation agent containing 10% by volume of Triton X-100, 0.5% by volume of sodium chloride, 20 μg / mL of heparin solution, 5% by volume of donkey serum, 15% by volume of glycerol, 1% by volume of 2,6-di-O-methyl-β-cyclodextrin, 5% by volume of N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, 2 mol / L of urea, and 50 mmol / L of glycine, and adjust the pH value to 7.2.

[0085] (3) Reagent C: Using PBS solution as solvent, prepare a second incubation agent containing 0.5% by volume of Triton X-100, 20 μg / mL of heparin solution, 0.5% by volume of sodium chloride, 10% by volume of glycerol, 1% by volume of 2,6-di-O-methyl-β-cyclodextrin, and 50 mmol / L of glycine, and adjust the pH value to 7.2.

[0086] (4) Reagent D: Using PBS solution as solvent, prepare a cleaning solution containing 0.2% by volume of Triton X-100 and 20 μg / mL of heparin solution, and add 20% by volume of glycerol to reagent D to prepare reagent D2.

[0087] (5) Reagent E: Using benzyl ether as solvent, prepare a refractive index matching agent containing 1% by volume of vitamin E.

[0088] Comparative Example 1: Preparation of a biological tissue clearing kit

[0089] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent A does not contain 2,6-di-O-methyl-β-cyclodextrin.

[0090] Comparative Example 2: Preparation of a biological tissue clearing kit

[0091] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent B does not contain 2,6-di-O-methyl-β-cyclodextrin.

[0092] Comparative Example 3: Preparation of a biological tissue clearing kit

[0093] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent B does not contain glycerol.

[0094] Comparative Example 4: Preparation of a biological tissue clearing kit

[0095] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent B does not contain N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine.

[0096] Comparative Example 5: Preparation of a biological tissue clearing kit

[0097] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent B does not contain urea.

[0098] Comparative Example 6: Preparation of a biological tissue clearing kit

[0099] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent C does not contain 2,6-di-O-methyl-β-cyclodextrin.

[0100] Comparative Example 7: Preparation of a biological tissue clearing kit

[0101] Compared with Example 1, the biological tissue clearing kit of this comparative example is different only in that reagent C does not contain glycerol.

[0102] Experimental Example 1: Vascular clearing imaging of the entire knee joint of mice

[0103] In this test example, the biological tissue clearing kit configured in Example 1 was used to process the biological tissue.

[0104] (1) Cleaning, fixing, and separating biological tissues: Weigh the mice (12 weeks old, C57BL / 6 mice) (body weight about 25g), and use 0.9% saline to prepare a mixed anesthetic (50mg / kg ketamine + 2.5mg / kg xylazine). After the mice were anesthetized by intraperitoneal injection, the chest cavity of the mice was opened to expose the heart. The needle of the infusion set was inserted into the left ventricle through the apex of the heart, and a 10ml syringe was used to infuse 50 units of heparin solution precooled at 4°C in advance, and the axillary vein was quickly cut to flush the blood. After flushing, a 30ml syringe was used to infuse PBS solution precooled at 4°C in advance until the muscle, liver, and spleen tissues turned white, followed by infusion of 30ml of paraformaldehyde solution (4% by mass volume) to further flush and fix the tissues. After the infusion was completed, the right knee joint of the mouse was removed.

[0105] (2) Decalcification: The right knee joint of the mouse was immersed in a paraformaldehyde solution (1% by mass volume) and fixed on a shaker at 4°C for 12 hours. The fixative was then discarded and the tissue was rinsed three times with a PBS solution, each for 1 hour. A 350 mmol / L ethylenediaminetetraacetic acid solution was prepared and its pH was adjusted to 6.5. The tissue was immersed in the ethylenediaminetetraacetic acid solution and placed on a shaker at 37°C for 4 days for decalcification, with the solution replaced every 24 hours.

[0106] (3) Antigen repair: Use ultrapure water to prepare an antigen repair solution containing 0.01 mol / L sodium citrate and 0.1% Tween-20 by volume. Soak the tissue in the antigen repair solution and place it in a 95°C incubator for 20 minutes for antigen repair.

[0107] (4) Dehydration, degreasing, and decolorization: After repair, use ultrapure water to dilute the methanol solution and prepare methanol solutions with volume fractions of 20%, 40%, 60%, 80%, 100%, and 100%, respectively. Dehydrate the tissues in the order of increasing methanol concentration gradient on a shaker at room temperature, and treat each solution for 2 hours. Then prepare a mixed degreasing solution with a volume ratio of dichloromethane to methanol of 2:1 to degrease the tissue for 12 hours, and replace the fresh degreasing solution every 4 hours. After degreasing, use methanol to wash twice, each time for 2 hours. Prepare a mixed decolorizing solution with hydrogen peroxide (volume fraction of 30%) and methanol in a volume ratio of 1:9, and immerse the tissue in the mixed decolorizing solution and place it on a shaker at 4°C for decolorization for 12 hours.

[0108] (5) Permeabilization, antibody incubation and washing: (a) Treat the tissue with 2 mL of reagent A in a 37°C shaker for 12 hours. Use ultrapure water to dilute acetone to prepare acetone solutions with volume fractions of 20%, 40%, 60%, 80%, 100%, and 100%, respectively. Dehydrate the tissue in the order of increasing concentration gradient, and the dehydration time for each step is 2 hours. After dehydration, evaporate and dry in a negative pressure device for 40 minutes to obtain dried right knee joint tissue. (b) Primary antibody incubation: Use reagent B as solvent, prepare 120 μL of antibody preliminary incubation solution at a dilution ratio of 20:1 between reagent B and Anti-CD31 antibody (R&D system, catalog number: AF3628), and use the antibody preliminary incubation solution to treat the dried tissue in a negative pressure device for 4 hours. After the treatment is completed, add reagent C to the preliminary incubated tissue to 1 ml, and perform a final incubation for 12 hours in a shaker at room temperature. After incubation, the tissue was washed alternately with reagent D and reagent D2 for 48 hours, and the reagents were replaced every 12 hours. After washing, the tissue was dehydrated in 20%, 40%, 60%, 80%, 100%, and 100% acetone solutions, with each dehydration time of 2 hours. After dehydration, the tissue was placed in a negative pressure device for evaporation and drying for 40 minutes. (c) Secondary antibody incubation: Reagent B was used as the solvent, and reagent B was mixed with antibody Donkey Anti-Goat IgG H&L (Alexa Fluor 647) (Abcam, catalog number: ab150135) was diluted with 120 μL of antibody initial incubation solution at a volume ratio of 40:1, and the tissue was treated with the antibody initial incubation solution in a negative pressure device for 4 hours. Reagent C was added to 1 ml for a final incubation at room temperature for 12 hours. After the incubation was completed, the tissue was alternately washed with reagent D and reagent D2 (the amount of reagent D and reagent D2 was 7 mL each time) for 48 hours, and replaced every 12 hours.

[0109] (6) Dehydration, transparentization and imaging: Use ultrapure water to dilute the methanol solution and prepare methanol solutions with volume fractions of 20%, 40%, 60%, 80%, 100%, and 100%. Dehydrate the tissues in the order of increasing methanol concentration gradient on a room temperature shaker, and treat each solution for 2 hours. After dehydration, wash the tissues with dichloromethane 4 times, 30 minutes each time. Use reagent E to match the refractive index of the tissue on a room temperature shaker for 48 hours, and replace fresh reagent every 12 hours. After the refractive index matching is completed, use a wide-field fluorescence microscope, a laser sheet scanning microscope, a laser confocal microscope, or a two-photon fluorescence microscope to scan and image it. After the refractive index matching is completed, the tissue can be immersed in reagent E and stored in the dark at 4°C for several weeks.

[0110] Experimental Example 2: Vascular clearing imaging of the entire knee joint of mice

[0111] In this test example, the biological tissue clearing kit configured in Example 1 was used to process the biological tissue.

[0112] Compared with Experimental Example 1, the tissue clearing method of this Experimental Example is different only in that: the antibody used in the primary antibody incubation in step (ii) of step (5) is Anti-CGRP antibody (Abcam, Catalog No.: ab47027), and the antibody used in the secondary antibody incubation in step (iii) is Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 555 (Thermo Fisher, Cat. No. A31572).

[0113] like Figure 5 and Figure 6 As shown, the use of the biological tissue clearing kit and the biological tissue clearing imaging method of the present invention can make the right knee joint tissue of the mouse quickly clear, and the cleared tissue can present a complete three-dimensional image using a microscope. It can be seen that the transparent tissue obtained by the tissue clearing reagent and the tissue clearing imaging method of the present invention can fully meet the needs of three-dimensional imaging.

[0114] Test Example 3:

[0115] In this test example, the biological tissue clearing kit configured in Example 1 and Comparative Examples 1-7 was used to process the biological tissue, and the same tissue clearing imaging method as in Test Example 1 was used.

[0116] Calculation method for tissue labeling percentage: V 标记 (Volume of tissue successfully cleared) / V 总 (total tissue volume).

[0117] like Figure 7 As shown, the use of the biological tissue clearing kit of the present invention can achieve nearly 100% transparent marking effect on biological tissues. When any important component is missing in the biological tissue clearing kit, it is impossible to comprehensively and effectively mark the target tissue structure. Therefore, the composition of the biological tissue clearing kit of the present invention is crucial to achieving efficient and high-quality clearing and marking effects.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A biological tissue clearing kit, characterized in that: It includes a biological tissue permeabilizing agent and an antibody incubating agent; the antibody incubating agent includes a first incubating agent and a second incubating agent; the biological tissue permeabilizing agent or the second incubating agent includes 2,6-di-O-methyl-β-cyclodextrin; the first incubating agent includes 2,6-di-O-methyl-β-cyclodextrin, N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine and urea.

2. The biological tissue clearing kit according to claim 1, characterized in that: It also includes a cleaning agent and a refractive index matching agent; the cleaning agent includes a non-ionic surfactant with a volume percentage of 0.01%-0.2% and a heparin solution with a concentration of 5μg / mL-20μg / mL; the refractive index matching agent includes an antioxidant with a volume percentage of 0.1%-1%.

3. The biological tissue clearing kit according to claim 2, characterized in that: The cleaning agent also includes glycerin in a volume percentage of 2.5%-20%.

4. The biological tissue clearing kit according to claim 1, characterized in that: The biological tissue permeabilizing agent includes 0.1%-1% of 2,6-di-O-methyl-β-cyclodextrin by mass volume; the first incubating agent includes 0.1%-1% of 2,6-di-O-methyl-β-cyclodextrin by mass volume, 2.5%-5% of N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine by volume, and 0.5mol / L-2mol / L of urea; the second incubating agent includes 0.1%-1% of 2,6-di-O-methyl-β-cyclodextrin by mass volume.

5. The biological tissue clearing kit according to claim 4, characterized in that: The biological tissue permeabilizing agent includes 1% by mass volume of 2,6-di-O-methyl-β-cyclodextrin; the first incubating agent includes 1% by mass volume of 2,6-di-O-methyl-β-cyclodextrin, 2.5% by volume of N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, and urea with a concentration of 1 mol / L; the second incubating agent includes 1% by mass volume of 2,6-di-O-methyl-β-cyclodextrin.

6. The biological tissue clearing kit according to claim 1, characterized in that: The first incubation agent or the second incubation agent further comprises glycerol in a volume percentage of 2.5%-20%.

7. Use of the biological tissue clearing kit according to any one of claims 1 to 6 in biological tissue clearing imaging.

8. The use according to claim 7, characterized in that: The biological tissue is any one of vascular tissue, neural tissue, lymphatic vessel tissue, muscle tissue, and osteochondral tissue of the musculoskeletal system.

9. A biological tissue transparent imaging method, characterized in that: The following steps are involved: (i) Cleaning and fixing biological tissues; performing antigen repair; and then dehydrating, degreasing and decolorizing the tissues; (ii) using the biological tissue permeabilization agent according to any one of claims 1 to 6 to permeabilize the tissue, dehydrate and dry it to obtain a dried tissue; (iii) using the first incubation agent described in any one of claims 1 to 6 to dilute the antibody to obtain a preliminary antibody incubation solution, incubating the tissue for 2h-24h, adding the second incubation agent described in any one of claims 1 to 6, and continuing to incubate for 6h-24h; washing, dehydrating, and imaging.

10. The biological tissue clearing imaging method according to claim 9, characterized in that: In the steps (iii) and / or (iv), the cleaning is performed by alternately using the cleaning agent described in claim 2 and the cleaning agent described in claim 3.

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