A clinical and animal tissue rapid transparent reagent and three-dimensional imaging method based on DOC-chaps cooperation

Abstract

The application discloses a kind of DOC-Chaps synergistic core's clinical and animal tissue rapid transparent reagent and three-dimensional imaging method. Rapid transparent reagent is mainly dissolved in deionized water by sodium deoxycholate (DOC), 3-[(3-cholamide propyl) dimethylamino]-1-propane sulfonic acid (Chaps), urea and 1-methylimidazole; DOC efficiently dissolves high-density lipid, Chaps gently destroys membrane structure, both complement each other, realize lipid rapid, depth clearance and significantly reduce light scattering; urea loosens collagen fiber and protein aggregate, improves the penetration rate of reagent; 1-methylimidazole selectively removes hemoglobin, reduces light absorption. The transparent reagent of the application can make millimeter thick brain slices in 4 (mouse brain) / 6 (human brain) hours The light transmittance of specific wavelength is greater than or equal to 90%, and there is no obvious tissue shrinkage / expansion, the compatibility of fluorescent protein and antibody label is excellent, suitable for three-dimensional imaging of high-lipid, high-hemoglobin samples such as in-vitro human brain, kidney, liver and mouse brain, heart, kidney, liver, intestinal tract, embryo, etc.

Description

Technical Field

[0001] This invention belongs to the field of biological sample and tissue processing, and relates to the field of biomedical engineering. Specifically, it relates to a rapid clearing reagent and three-dimensional imaging method for clinical and animal tissues based on DOC-Chaps synergy. Background Technology

[0002] Tissue optical transparency technology uses chemical reagents to reduce the differences in light scattering and absorption within tissues, allowing light to penetrate centimeter-thick sections and achieve three-dimensional overall imaging, avoiding information loss caused by traditional sectioning. Current optical transparency technologies are mainly divided into three categories:

[0003] 1) Solvent-based (such as DISCO series): Dehydrates and degreases with organic solvents, resulting in rapid transparency, but easily causes fluorescence quenching;

[0004] 2) Water-soluble type (such as CUBIC series): Uses mild components such as urea and glycerin, which have good fluorescence retention, but have the disadvantages of low degreasing efficiency and long time consumption;

[0005] 3) Hydrogel type (such as CLARITY series): The structure is stable by cross-linking proteins with acrylamide. However, the dense gel restricts antibody penetration and is prone to causing tissue swelling and brittleness.

[0006] It is worth noting that all the water-soluble solutions mentioned above are based on nonionic or zwitterionic detergents (Triton X-100, Teeen 20, Chaps, etc.). Strong anionic detergents such as DOC (sodium deoxycholate) have long been avoided in this field because they are believed to severely damage tissue structure. Therefore, how to utilize the high-efficiency degreasing ability of DOC to overcome the current bottleneck in transparency efficiency while maintaining tissue integrity and fluorescence signal remains an unresolved technological gap. Summary of the Invention

[0007] To address the problems existing in the background technology, this invention, for the first time, synergistically introduces the strong anionic detergent DOC and the zwitterionic detergent Chaps into a water-based clearing system, pioneering a "DOC-Chaps complementary degreasing" strategy. Due to its small charge, strong hydrophobicity, and negative charge, DOC can penetrate millimeter-scale tissues in a short time under the influence of an electric field, first efficiently dissolving myelin sheaths and highly hydrophobic lipids and loosening heme-protein complexes. Subsequently, the zwitterionic detergent Chaps gently removes residual phospholipid bilayers and membrane proteins, protecting antigenic epitopes and preventing tissue fragmentation. Simultaneously, urea dissociates the lipoprotein network, and 1-methylimidazole removes heme. Both processes are accelerated by the pre-exposure effect of DOC-Chaps, shortening the overall clearing time. The clearing reagent of this invention can achieve a transmittance of ≥90% at a specific wavelength for millimeter-thick brain slices within 4 / 6 hours, with no significant tissue shrinkage / swelling, achieving rapid and deep clearing of high-lipid, heme-rich tissues.

[0008] This invention fills the technological gap in the use of "DOC-Chaps" amphoteric detergent combination for water-based transparency of biological tissues, and provides a brand-new technical solution for three-dimensional imaging of biological samples at the millimeter to centimeter level.

[0009] The technical solution adopted in this invention is:

[0010] I. DOC-Chaps Synergistic Rapid Tissue Clearing Reagent for Clinical and Animal Use

[0011] The rapid clearing reagent for clinical and animal tissues mainly consists of sodium deoxycholate, 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid, urea, and 1-methylimidazole dissolved in deionized water. In this reagent, sodium deoxycholate and 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid form an anionic-zwitterionic synergistic system, which is used to efficiently and synergistically dissolve lipids and cleave heme-protein complexes in animal tissues with minimal deformation.

[0012] The sodium deoxycholate in the aforementioned rapid clearing reagent for clinical and animal tissues has a mass concentration of 0.5-2%. The 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid in the aforementioned rapid clearing reagent for clinical and animal tissues has a mass concentration of 2-10%. The urea in the aforementioned rapid clearing reagent for clinical and animal tissues has a molar concentration of 4-8 M. The 1-methylimidazole in the aforementioned rapid clearing reagent for clinical and animal tissues has a molar concentration of 50-200 mM.

[0013] Preferably, for high-fat tissues (such as the brain, high-fat tissues), the concentrations of DOC, Chaps, urea, and 1-methylimidazole are 0.5-2%, 1-2%, 4-6 M, and 50-100 mM, respectively. Preferably, for heme-rich tissues (such as the kidneys, liver, etc.), the concentrations of DOC, Chaps, urea, and 1-methylimidazole are 0.5-1%, 2-5%, 6-8 M, and 100-200 mM, respectively.

[0014] The DOC (415 Da) anionic detergent can rapidly penetrate deep into tissues, efficiently dissolve highly hydrophobic lipids, and cleave heme-protein complexes, serving as a "pre-resin" for subsequent steps. The Chaps (615 Da) zwitterionic detergent gently disrupts the phospholipid bilayer while preserving antigenic epitopes; it forms a "powerful-mild" complementary pair with DOC, synergistically removing residual lipids and significantly reducing light scattering. The urea-denatured lipoprotein network further releases membrane-bound lipids, enhancing the DOC / Chaps penetration rate. The 1-methylimidazole catalyzes lipid saponification and heme degradation, reducing light absorption while simultaneously enhancing DOC / Chaps activity.

[0015] II. Application of DOC-Chaps-based rapid tissue clearing reagents in clinical and animal settings

[0016] Applications of transparent imaging of clinical animal tissues. The clinical and animal tissues referred to are isolated human brains, kidneys, livers, or mammalian / reptilian brains, hearts, kidneys, livers, intestines, and embryos.

[0017] III. Rapid Clearing Reagents for Clinical and Animal Tissues: A Rapid Clearing and Imaging Method Based on DOC-Chaps Synergy for Ex vivo Clinical Samples or Animal Tissues

[0018] After fixing the isolated biological tissue, the fixed tissue was directly immersed in the rapid clearing reagent and left to stand at 37°C until the fixed tissue became completely transparent, thus obtaining a clear tissue. The clear tissue was then processed to obtain a processed tissue, and finally, a three-dimensional image of the processed tissue was performed.

[0019] After adding sodium deoxycholate, 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid, urea, and 1-methylimidazole to deionized water and stirring until completely dissolved, the above-mentioned rapid clearing reagent for clinical and animal tissues is obtained. "Rapid" in "rapid clearing" refers to a treatment time of ≤4 h for mouse brains and ≤6 h for human brains.

[0020] The process involves processing the transparent tissue to obtain the processed tissue, and finally performing three-dimensional imaging on the processed tissue. Specifically, the transparent tissue is stained and subjected to refractive index matching operations to obtain the processed tissue. The processed tissue is then subjected to three-dimensional imaging of biological samples at the millimeter to centimeter scale using methods such as laser confocal microscopy, two-photon microscopy, large tissue light film, or wide-field fluorescence microscopy. This is the imaging method for transparent tissue.

[0021] Here, "millimeter-level" refers to the rapid transparency of small tissues achieved in a short time, while "centimeter-level" refers to the transparency of large tissues at the centimeter level, which can be achieved by extending the transparency time. The staining treatment is one of immunofluorescence, immunohistochemistry, chemical dyes, or viral fluorescent protein labeling.

[0022] The specific steps are as follows:

[0023] 1) Obtaining and fixing biological tissues

[0024] Isolated human brain, kidney, liver, or brain, heart, kidney, liver, intestine, and embryo from mammals / reptiles were perfused and fixed with 4% PFA.

[0025] 2) DOC-Chaps Collaborative Transparency

[0026] The fixed tissue was directly immersed in the reagent of the present invention (DOC-Chaps-urea-1-methylimidazole) and allowed to stand at 37°C until it became completely transparent (usually ≤6 h).

[0027] 3) Staining and Imaging

[0028] After transparency, immunofluorescence, immunohistochemistry, and chemical dye or viral fluorescent protein labeling are performed sequentially. After refractive index matching, three-dimensional imaging is completed using laser confocal microscopy, two-photon microscopy, light sheet microscopy, or wide-field fluorescence microscopy.

[0029] This "DOC-Chaps synergistic strategy" introduces sodium deoxycholate (DOC), a strong anionic detergent, into a water-based transparency system for the first time. As part of the water-based transparency formulation, it synergizes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid (Chaps) to form a "DOC+Chaps" two-component core formulation, which takes into account speed, depth and structural integrity, and breaks through the technical prejudice of avoiding DOC in the field for a long time.

[0030] Among them, DOC, with its strong hydrophobic properties, efficiently dissolves highly dense lipids, while Chaps gently disrupts the membrane structure. The two components complement each other synergistically, achieving rapid and deep lipid removal and significantly reducing light scattering. Urea loosens collagen fibers and protein aggregates, enhancing reagent penetration. 1-Methylimidazole selectively removes heme, reducing light absorption. This invention is suitable for three-dimensional imaging of high-lipid, high-heme samples from human brain, kidney, liver, and mouse brain, heart, kidney, liver, intestines, and embryos.

[0031] The beneficial effects of this invention are:

[0032] 1) The clearing reagents used in this invention are all commercially available products, and the preparation process is simple and convenient, allowing for immediate use. They are compatible with various staining methods such as immunofluorescence, chemical dyes, and viral labeling, and are suitable for high-lipid / high-heme samples.

[0033] 2) The clearing reagent of the present invention can be selected according to the differences in tissue samples, and different reagent types and concentrations can be selected. The clearing reagent of the present invention is suitable for traditionally difficult-to-clear samples such as isolated human brain, liver, and kidney, and the sample tissue hardly undergoes any deformation (expansion or contraction) and the structure remains intact. The clearing reagent of the present invention adopts a gentle degreasing strategy, which hardly causes tissue deformation and completely preserves the fine structure and antigen epitopes.

[0034] 3) Compared with existing technologies, the clearing agent of this invention, through the gradient synergistic effect of its components, can achieve a light transmittance of ≥90% at a specific wavelength for millimeter-thick tissues within 4-6 hours, without significant tissue shrinkage / swelling, which is significantly faster than existing water-soluble types. The clearing process of this invention can shorten the clearing treatment time, improve the clearing effect, and does not produce significant deformation.

[0035] 4) Compared with the prior art, the clearing reagent of the present invention is compatible with a variety of staining methods and is fluorescence-friendly. It has excellent compatibility between fluorescent protein and antibody labeling, and both fluorescent protein and antibody labeling maintain high fidelity.

[0036] 5) This invention is the first to introduce DOC into a water-based transparentization system for biological tissues, forming a DOC-Chaps synergistic degreasing strategy, filling a technological gap. Attached Figure Description

[0037] Figure 1 This is a comparison of the clearing effects of three different clearing agents on 1000 μm mouse brain tissue. In the figure, a, b, and c represent before treatment; d, e, and f represent after treatment.

[0038] Figure 2 This is a comparison of the transmittance of three groups of mouse brain tissues treated with different clearing agents in the wavelength range of 400-800 nm.

[0039] Figure 3The image shows a comparison of the imaging effects of three groups of mouse brain tissue labeled with Anti-Iba1 antibody at Scale bar-150 μm. In the figure, a, b, and c represent Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0040] Figure 4 This is a comparison of the transparency effects of three different clearing agents on 1000 μm clinical human brain tissue. In the figure, a, b, and c represent before treatment; d, e, and f represent after treatment.

[0041] Figure 5 This is a comparison of the transmittance of three groups of clinical human brain tissues treated with different clearing agents in the wavelength range of 400-800 nm.

[0042] Figure 6 The image shows a comparison of the imaging effects of three groups of clinical human brain tissue labeled with Anti-Podxl antibody at Scalebar-100 μm. In the figure, a, b, and c represent Example 2, Comparative Example 3, and Comparative Example 4, respectively.

[0043] Figure 7 These are comparative images of the transparency effects of four different transparentizing reagents on 1000 μm mouse brain tissue. In the images, a, b, c, and d are images of mouse brain tissue before treatment, and e, f, g, and h are images of mouse brain tissue after treatment. In the images, i, j, k, and l are comparison images of mouse brain tissue before and after transparentization, and m, n, o, and p are comparison images of the imaging effects. Scale bar-150 μm. Detailed Implementation

[0044] The present invention will be described in detail below with reference to specific implementation examples. These examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0045] The core of this invention lies in the first-ever construction of a "DOC-Chaps synergistic water-based transparentization system." DOC (415 Da), as a strong anionic detergent, first penetrates the tissue, efficiently dissolving highly hydrophobic lipids and loosening heme-protein complexes. Chaps (zwitterionic) then gently disrupts the phospholipid bilayer, compensating for DOC's insufficient effect on polar lipids, achieving a complementary "powerful-gentle" degreasing process. Urea simultaneously dissociates the lipid-protein network and reduces tissue density, while 1-methylimidazole catalyzes heme degradation and improves optical homogeneity. These four components work in a stepwise manner—penetration-degreasing-dissociation-homogenization—achieving millimeter-level tissue transparentization in a short time (4 hours for mouse brain or 6 hours for human brain), while maintaining structural integrity, fluorescence fidelity, and antibody penetration. This provides a novel solution for three-dimensional overall imaging of high-lipid and high-heme samples.

[0046] All reagents involved in this invention are commercially available and are not subject to any special restrictions.

[0047] The biological tissues include, but are not limited to, isolated human clinical samples and mouse brain, kidney, liver, heart, pancreas, and intestine.

[0048] The biological tissue can be a sheet-like sample (of varying thickness) or a complete animal organ.

[0049] The specific implementation steps of this invention are as follows:

[0050] 1) Obtaining and fixing isolated biological tissues;

[0051] 2) Processing and sectioning biological tissues;

[0052] 3) Prepare different clearing reagents according to the sample and incubate the tissue sample;

[0053] 4) Clean the tissue samples and incubate them with the first and second antibodies respectively. Before changing the second antibody, the samples need to be cleaned.

[0054] 5) After staining, clean the sample, perform refractive index matching, and image it in a transparent state.

[0055] Imaging microscopes include, but are not limited to, laser confocal microscopes, two-photon fluorescence microscopes, large tissue light slide microscopes, and wide-field fluorescence microscopes.

[0056] The steps of the embodiments of the present invention are as follows:

[0057] Example 1: Obtaining mouse brain slices and performing clearing and staining.

[0058] All animal experiments involved in this invention were conducted in accordance with the guidelines of the Animal Ethics Committee of Zhejiang University, China.

[0059] The specific steps are as follows: Adult mice were deeply anesthetized (8 weeks) with 1% sodium pentobarbital. The thoracic cavity was opened, and 40 ml of ice-cold PBS (phosphate-buffered saline) was perfused through the heart to flush out the blood. Then, 30 ml of 4% polytetrafluoroethylene (PFA) was perfused for fixation. The mouse brain was removed and placed in a test tube containing 4% PFA, and stored overnight at 4 ℃. The brain was then sectioned using a shaker.

[0060] 1000 μm sections of mouse brain were obtained, washed with PBS, and photographed. (Photos are shown below.) Figure 1As shown in Figure a. The tissue sections were placed in 24-well plates and treated with the above-prepared clearing reagent (5% Chaps, 2% DOC, 5 M urea, 200 mM 1-methylimidazole) for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R (antipyrine, nicotinamide, and N-butyldiethanolamine dissolved in water at a mass-to-volume ratio of 45%, 30%, and 0.5%, respectively) refractive index matching solution until the tissue was completely clear. The sections were then photographed, as shown in Figure a. Figure 1 As shown in d. The transmittance of the tissue sections was then measured using a UV-2600 ultraviolet spectrophotometer, covering the commonly used laser range (400-800 nm). The test results are shown in Figure d. Figure 2 The blue markers indicate (n=3).

[0061] After data collection, the samples were washed again with PBS. The cleared mouse brain tissue was then incubated for 48 hours in a staining buffer containing 1:200 Anti-Iba1 primary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After incubation, the tissue was washed three times with PBS for one hour each time, followed by incubation for 24 hours with the corresponding secondary antibody buffer containing 1:200 secondary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After this incubation, the tissue sections were washed three times with PBS for one hour each time. Following washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The tissue was then imaged using a laser confocal microscope. The results are shown below. Figure 3 As shown in a.

[0062] Comparative Example 1: Obtain mouse brain slices and perform clearing and staining.

[0063] 1000 μm sections of mouse brain were obtained, washed with PBS, and photographed. (Photos are shown below.) Figure 1 As shown in b. The tissue sections were placed in 24-well plates and treated with the above-prepared clearing reagent (0% Chaps, 2% DOC, 5 M urea, 200 mM 1-methylimidazole) for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photographs are shown in Figure b. Figure 1 As shown in e. The transmittance of the tissue sections was then tested using a UV spectrophotometer, with test wavelengths covering the commonly used laser range. The test results are shown in... Figure 2 The red markings indicate (n=3).

[0064] After data collection, the samples were washed again with PBS. The cleared mouse brain tissue was then incubated for 48 hours in a staining buffer containing 1:200 Anti-Iba1 primary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After incubation, the tissue was washed three times with PBS for one hour each time, followed by incubation for 24 hours with the corresponding secondary antibody buffer containing 1:200 secondary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After this incubation, the tissue sections were washed three times with PBS for one hour each time. Following washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The tissue was then imaged using a laser confocal microscope. The results are shown below. Figure 3 As shown in b.

[0065] Comparative Example 2: Obtaining mouse brain slices and performing clearing and staining.

[0066] 1000 μm sections of mouse brain were obtained, washed with PBS, and photographed. (Photos are shown below.) Figure 1 As shown in c. The tissue sections were placed in 24-well plates and treated with the above-prepared clearing reagent (5% Chaps, 0% DOC, 5 M urea, 200 mM 1-methylimidazole) for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photograph is shown in [image missing]. Figure 1 As shown in f. Then, a UV spectrophotometer was used to test the transmittance of the tissue sections. The test wavelengths covered the commonly used laser range, and the test results are shown in f. Figure 2 The green marker indicates (n=3).

[0067] After data collection, the samples were washed again with PBS. The cleared mouse brain tissue was then incubated for 48 hours in a staining buffer containing 1:200 Anti-Iba1 primary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After incubation, the tissue was washed three times with PBS for one hour each time, followed by incubation for 24 hours with the corresponding secondary antibody buffer containing 1:200 secondary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After this incubation, the tissue sections were washed three times with PBS for one hour each time. Following washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The tissue was then imaged using a laser confocal microscope. The results are shown below. Figure 3 As shown in c.

[0068] Example 2: Obtaining human brain slices and performing clearing and staining.

[0069] The clinical human brain tissue used in the experiments was obtained from the Chinese Brain Bank of Zhejiang University School of Medicine.

[0070] The specific steps are as follows: The obtained clinical human brain tissue was sectioned by shaking; the obtained 1000 μm sections were washed with PBS and photographed, as shown in the image. Figure 4 As shown in Figure a. The tissue sections were placed in 24-well plates and treated with the clearing reagent (5% Chaps, 2% DOC, 5 M urea, 200 mM 1-methylimidazole) prepared above for 6 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photographs are shown in Figure a. Figure 4 As shown in d. The transmittance of the tissue sections was then measured using a UV spectrophotometer, with the test wavelengths covering the commonly used laser range. The test results are shown in Figure d. Figure 5 The blue markers indicate (n=3).

[0071] After data collection, the samples were washed again with PBS. The cleared human brain tissue was then incubated for 48 hours in a staining buffer containing 1:200 Anti-Podxl primary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After incubation, the tissue was washed three times with PBS for one hour each time, followed by incubation for 24 hours with the corresponding secondary antibody buffer containing 1:200 secondary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After this incubation, the tissue sections were washed three times with PBS for one hour each time. Following washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The tissue was then imaged using a laser confocal microscope. The results are shown below. Figure 6 As shown in a.

[0072] Comparative Example 3: Obtaining human brain slices and performing clearing and staining.

[0073] The specific steps are as follows: The obtained clinical human brain tissue was sectioned by shaking; the obtained 1000 μm sections were washed with PBS and photographed, as shown in the image. Figure 4 As shown in b. The tissue sections were placed in 24-well plates and treated with the above-prepared clearing reagent (0% Chaps, 2% DOC, 5 M urea, 200 mM 1-methylimidazole) for 6 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photographs are shown in Figure b. Figure 4 As shown in e. The transmittance of the tissue sections was then tested using a UV spectrophotometer, with test wavelengths covering the commonly used laser range. The test results are shown in... Figure 5 The red markings indicate (n=3).

[0074] After data collection, the samples were washed again with PBS. The cleared human brain tissue was then incubated for 48 hours in a staining buffer containing 1:200 Anti-Podxl primary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After incubation, the tissue was washed three times with PBS for one hour each time, followed by incubation for 24 hours with the corresponding secondary antibody buffer containing 1:200 secondary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After this incubation, the tissue sections were washed three times with PBS for one hour each time. Following washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The tissue was then imaged using a laser confocal microscope. The results are shown below. Figure 6 As shown in b.

[0075] Comparative Example 4: Obtaining human brain slices and performing clearing and staining.

[0076] The specific steps are as follows: The obtained clinical human brain tissue was sectioned by shaking; the obtained 1000 μm sections were washed with PBS and photographed, as shown in the image. Figure 4 As shown in c. The tissue sections were placed in 24-well plates and treated with the clearing reagent prepared above (5% Chaps, 0% DOC, 5 M urea, 200 mM 1-methylimidazole) for 6 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photograph is shown in c. Figure 4 As shown in f. Then, a UV spectrophotometer was used to test the transmittance of the tissue sections. The test wavelengths covered the commonly used laser range, and the test results are shown in f. Figure 5 The green marker indicates (n=3).

[0077] After data collection, the samples were washed again with PBS. The cleared human brain tissue was then incubated for 48 hours in a staining buffer containing 1:200 Anti-Podxl primary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After incubation, the tissue was washed three times with PBS for one hour each time, followed by incubation for 24 hours with the corresponding secondary antibody buffer containing 1:200 secondary antibody, 1% donkey serum, and 3% DMSO dissolved in PBS. After this incubation, the tissue sections were washed three times with PBS for one hour each time. Following washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The tissue was then imaged using a laser confocal microscope. The results are shown below. Figure 6 As shown in c.

[0078] Depend on Figures 1-3The corresponding transmittance data show that only when DOC and Chaps are present simultaneously can a 1000 μm mouse brain slice achieve a transmittance of over 90% within 4 hours, with intact tissue structure and clear fluorescence signal. The group lacking DOC (Comparative Example 2) had a transmittance of less than 60%, while the group lacking Chaps (Comparative Example 1) showed some tissue fragmentation and significantly insufficient transmittance. This result directly proves that the addition of DOC is not a simple replacement or concentration increase, but rather forms a synergistic "complementary lipid-lowering channel" with Chaps. This combination constitutes the core innovation of this invention.

[0079] Example 3: Obtaining mouse brain slices and performing clearing and staining.

[0080] 1000 μm sections of mouse brain were obtained, washed with PBS, and photographed. (Photos are shown below.) Figure 7 As shown in Figure a. The tissue sections were placed in 24-well plates and treated with the clearing reagent (5% Chaps, 2% DOC, 5 M urea, 200 mM 1-methylimidazole) prepared above for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photographs are shown in Figure a. Figure 7 As shown in e, the outlines of the graphic before and after transparency are compared as follows: Figure 7 As shown in i. After washing with PBS, Anti-α-SMA-FITC staining was performed, and the imaging results are as follows. Figure 7 As shown in m.

[0081] Comparative Example 5: Obtaining mouse brain slices and performing clearing and staining.

[0082] As described in the comparative literature (Daisuke K. et al. ClearSee: a rapid optical clearingreagent for whole-plant fluorescence imaging, Development 142, 4168-4179 (2015)), 1000 μm sections of mouse brain were obtained using a clearing reagent (10% xylitol, 15% DOC, 25% urea) with added DOC detergent. These sections were then washed with PBS and photographed. The photographs are shown below. Figure 7 As shown in b. The tissue sections were placed in a 24-well plate and treated with the clearing reagent prepared above for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear. The images are shown below. Figure 7 As shown in f, the outlines of the graphic before and after transparency are compared as follows: Figure 7As shown in j. After washing with PBS, Anti-α-SMA-FITC staining was performed, and the imaging results are as follows. Figure 7 As shown in n.

[0083] Comparative Example 6: Obtain mouse brain slices and perform clearing and staining.

[0084] 1000 μm sections of mouse brain were obtained, washed with PBS, and photographed. (Photos are shown below.) Figure 7 As shown in c. The tissue sections were placed in a 24-well plate and treated with the clearing reagent (0% Chaps, 2% DOC) prepared above for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photograph is shown in [image not provided]. Figure 7 As shown in g, the outlines of the graphic before and after transparency are compared as follows: Figure 7 As shown in k. After washing with PBS, Anti-α-SMA-FITC staining was performed, and the imaging results are as follows. Figure 7 As shown in o.

[0085] Comparative Example 7: Obtaining mouse brain slices and performing clearing and staining.

[0086] 1000 μm sections of mouse brain were obtained, washed with PBS, and photographed. (Photos are shown below.) Figure 7 As shown in d. The tissue sections were placed in 24-well plates and treated with the clearing reagent (5% Chaps, 0% DOC) prepared above for 4 hours. The sections were washed three times with PBS for 1 hour each time. After washing, the tissue sections were placed in CUBI-R refractive index matching solution until the tissue was completely clear, and then photographed. The photographs are shown in d. Figure 7 As shown in h, the outlines of the graphics before and after transparency are compared as follows: Figure 7 As shown in Figure 1. After washing with PBS, Anti-α-SMA-FITC staining was performed, and the imaging results are as follows. Figure 7 As shown in p.

[0087] A comparison of the external contours of the images before and after transparency reveals that only the sample slices in this invention exhibit almost zero deformation. However, three other comparative examples show that the transparent sample slices all exhibit varying degrees of deformation, with the deformation reaching almost a quarter of the original slice area, causing significant damage to the original tissue morphology. Furthermore, observation of the imaging results (m, n, o, p) shows that all embodiments of this invention demonstrate significantly improved transparency compared to comparative examples 5, 6, and 7, and only the embodiments of this invention achieve full-thickness imaging.

[0088] In summary, this invention is the first to synergistically introduce the strong anionic detergent DOC and the zwitterionic detergent Chaps into a water-based clearing system, forming a dual mechanism of "highly efficient degreasing and gentle shape preservation." This significantly improves the transmittance of millimeter-sized mouse / human brain slices within 4 / 6 hours, with almost zero shrinkage and zero expansion. The addition of urea and 1-methylimidazole further accelerates lipid-heme clearance, significantly improving antibody penetration depth and signal uniformity. Comparative experiments confirm that this approach is significantly superior to existing water-soluble techniques in terms of clearing rate, tissue integrity, and imaging quality, providing a breakthrough tool for three-dimensional overall imaging of high-lipid and high-heme samples.

[0089] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A DOC-Chaps co-protocol based reagent for rapid tissue transparency in clinical and animal tissues, characterized in that: The clinical and animal tissue rapid transparent reagent is mainly composed of sodium deoxycholate, 3-[(3-cholamide propyl) dimethylamino]-1-propanesulfonic acid, urea and 1-methyl imidazole dissolved in deionized water; the mass concentration of sodium deoxycholate in the clinical and animal tissue rapid transparent reagent is 0.5-2%; the mass concentration of 3-[(3-cholamide propyl) dimethylamino]-1-propanesulfonic acid in the clinical and animal tissue rapid transparent reagent is 2-10%.

2. The DOC-Chaps co-cores based rapid clearing reagent for clinical and animal tissues according to claim 1, characterized in that: The molar concentration of urea in the clinical and animal tissue rapid transparent reagent is 4-8 M.

3. The DOC-Chaps co-cores based rapid clearing reagent for clinical and animal tissues according to claim 1, characterized in that: The molar concentration of 1-methyl imidazole in the clinical and animal tissue rapid transparent reagent is 50-200 mM.

4. The use of the DOC-Chaps synergistic core-based reagent for rapid clearing of clinical and animal tissues according to any one of claims 1-3, characterized in that: The application of the clinical and animal tissue rapid transparent reagent to the transparentization and imaging of clinical and animal tissues.

5. The use of DOC-Chaps co-cores based reagent for rapid clearing of clinical and animal tissues according to claim 4, characterized in that: The clinical and animal tissues are the brain, heart, kidney, liver, intestine and embryo of an isolated mammal or reptile.

6. The method for rapid transparentization and imaging of clinical or animal tissues by using the reagent of DOC-Chaps synergistic core according to any one of claims 1-3, wherein the method comprises the following steps: (1) the clinical or animal tissues are cut into slices with a thickness of 2-5 mm; (2) the slices are placed in the reagent of DOC-Chaps synergistic core, and the slices are soaked in the reagent for 1-5 min; (3) the slices are taken out and observed under a microscope. The fixed tissue is obtained by fixing the isolated biological tissue, the transparentized tissue is obtained by directly immersing the fixed tissue in the rapid transparent reagent and standing at 37 DEG C until the fixed tissue is completely transparent, the treated tissue is obtained by treating the transparentized tissue, and finally the three-dimensional imaging of the treated tissue is performed.

7. The rapid clearing tissue and imaging method of claim 6, wherein: The three-dimensional imaging of the treated tissue is performed by using the laser confocal, two-photon, large tissue light sheet or wide-field fluorescence microscope mode.

8. The rapid clearing tissue and imaging method of claim 7, wherein: The staining is one of immunofluorescence, immunohistochemistry, chemical dye and virus fluorescent protein labeling.

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