Methods for large tissue labeling, transparentizing and imaging using antibodies

Abstract

The present invention relates to methods for large tissue labeling, transparentizing and / or imaging using labeling agents (e.g., antibodies), as well as uses and products related thereto. The invention includes, inter alia, methods of preparing animal tissue for fluorescence microscopy, animal tissue obtainable by said methods, methods of analyzing said animal tissue, and methods for detecting transfers, analyzing the biological distribution of biopharmaceutical drugs, and analyzing the biological distribution of nanoparticles. The methods of making animal tissue according to the present invention encompass whole body marking, transparentizing and imaging methods. The method of the invention is advantageous in that: They allow for visualization of individual cells within mammalian tissue, including the entire mouse body or other large tissue, tumor metastasis at the single cell level, and visualization of the distribution of biopharmaceutical drugs (e.g., tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, tumor metastasis, the distribution of cancer targeted therapy antibodies in a whole animal (e.g., a complete mouse).

Description

Technical Field

[0001] The present invention relates to methods for labeling, clearing and / or imaging of large tissues using labeling agents (e.g., antibodies), as well as uses and products related thereto. The present invention particularly includes methods for preparing animal tissues for fluorescence microscopy, animal tissues obtainable by the methods, methods for analyzing the animal tissues, and methods for detecting metastasis, analyzing the biodistribution of biopharmaceutical drugs, and analyzing the biodistribution of nanoparticles. The methods for preparing animal tissues according to the present invention include whole-body labeling, clearing and imaging methods. The methods of the present invention have the advantage that, for example, they allow visualization of single cells within mammalian tissues (including the entire mouse body or other large tissues), tumor metastasis at the single cell level, and visualization of the distribution of biopharmaceutical drugs at the single cell level in the whole mouse using labeling agents (e.g., antibodies) (e.g., the distribution of cancer-targeted therapeutic antibodies in the whole animal (e.g., a complete mouse)). Background Art

[0002] More than a century of dedicated work has resulted in a detailed understanding of the gross anatomy of the human body and common model organisms, as well as detailed organizational maps of many individual organs. However, mapping the distribution, connectivity, and molecular composition of cell types throughout the body under given experimental conditions remains challenging. For example, although the nervous system is connected to every part of the mammalian body, we do not yet have a cellular-level atlas of the nervous system that reveals the complex relationships between organs and between organs and the central nervous system. 1-3 Furthermore, most methods for imaging neural or other cells in a whole-body context rely on genetically modified animals. 4,5 , which severely limits the flexibility of experimental design.

[0003] Generating new transgenic animals to map changes in the distribution of relevant proteins is often prohibitively expensive and time-consuming. However, such whole-body connectivity maps are needed to understand the functional interdependencies between organ systems and how a disease that starts in one part of the body affects others, such as during neurodegeneration or systemic inflammation.

[0004] Whole-body imaging can capture cellular information and provide comprehensive biological insights into healthy rodents. However, although mice are a commonly used animal model, we still lack basic information about their bodies, namely how various organs and tissue systems are organized in the whole mouse.

[0005] Recent transparency methods have enabled the complete tissue 6 , mouse tissue 7 and body 3、8-14 , large human organs 15 , and even human embryos 16Although antibody labeling and imaging have become possible, we still lack suitable, widely applicable labeling methods for the whole mouse body. Previous whole-body imaging methods, such as CUBIC, PACT, and uDISCO, enable whole-body imaging, but they rely on transgenic expression of fluorescent proteins in cell subsets, such as mice expressing Thy-1 EGFP in neurons. 17 vDISCO Method 5 The mice were labeled throughout the body using small antibodies called nanobodies (1 / 10 the size of IgG). Compared to the thousands of traditional, unconjugated antibodies developed over the past few decades, very few nanobodies work in a histological setting.

[0006] Although it can be passed through the mouse blood vessels 5 Cardiac pumping solutions enable uniform labeling of the entire body with small molecules (e.g., DNA labeling dyes) or nanobodies (e.g., as described in WO 2018 / 224289 A1), but this has proven difficult for standard IgG antibodies because 1) antibodies degrade and / or precipitate during perfusion, 2) they do not uniformly penetrate different tissue layers including muscle and bone, and 3) cell membranes are not maximally permeabilized, so antibodies cannot penetrate deeply into all tissues with different properties.

[0007] Therefore, further improved and more versatile methods are needed for the preparation and analysis of tissues including whole animals and large mammalian brains. Specifically, indirect immunolabeling of whole mice using primary and secondary conventional antibodies will be a particularly valuable approach for many biological applications, including whole-body mapping of target cells. Summary of the invention

[0008] Here, a new technique is presented that allows high-resolution 3D imaging of the peripheral nervous system (PNS), lymphatic system, and vascular system in whole animals (e.g., mice). The underlying technology, called wildDISCO (immunolabeling of wild-type mice with DISCO clearing), is a chemical method that enhances the penetration of standard labeling agents, such as antibodies (preferably >100 kDa, e.g., ~150 kDa in size), into the whole body of an animal (e.g., ~2 cm in thickness of a mouse body). This method allows cholesterol extraction for permeabilization, thereby ensuring uniform penetration and staining of tissues throughout the entire body of a mouse, such as muscle, bone, brain, and spinal cord. Combining whole-body antibody labeling with DISCO-based tissue clearing allows us to provide whole-body cell type and protein distribution maps with unprecedented ease and help to improve our understanding of biological systems.

[0009] wildDISCO can reveal integrated neuronal, vascular, and lymphatic networks. By using this technique, PNS innervation in most organs can be observed, including the heart, lungs, liver, kidneys, stomach, and intestines. In addition, the vagus nerve that innervates the gastrointestinal tract can also be visualized. By using this technique, it is also possible to present the unevenly distributed capillaries in the center of the intestinal villi and the region-specific three-dimensional villus lymphatic network. Surprisingly, it was found that the lymph nodes are innervated by a group of PNs with immunomodulatory potential. By using this technique, it is also possible to image organ-specific vascular patterns and the cortical capillary network that serves as the main support for multiple bones. Therefore, mapping the entire mouse body system can provide a roadmap for a variety of studies, including neural circuits, immunoregulation, and angiogenesis throughout the mammalian body.

[0010] The invention also allows unbiased imaging of transparent whole mouse bodies at cellular resolution, providing a comprehensive view of biological systems (neural or lymphatic) in health and disease. wildDISCO does not rely on transgenic expression of fluorescent proteins and allows uniform and simultaneous staining of structures throughout the mouse body using readily available IgG antibodies.

[0011] This invention provides a versatile method. The mouse head is a perfect example of the versatility of this method because it combines hard tissue (skull) and soft tissue (brain). Using wildDISCO, it is possible to map the lymphatic vessels in and around the brain parenchyma of an intact mouse head.

[0012] In summary, wildDISCO technology enables uniform and simultaneous antibody staining in large tissues such as the whole mouse body. Previously unavailable 3D anatomical information becomes possible (for example, with the help of VR visualization), allowing for a more comprehensive understanding of the occurrence, progression, and extent of pathology at the level of the entire mouse organism.

[0013] The inventors reasoned that imaging optically transparent tissues, including mice, could be used as a powerful preclinical approach, such as detecting fluorescently labeled cancer cells and / or therapeutic antibodies at cellular resolution within the intact body. Typically, fluorescent labeling of cancer cells in vitro or in vivo is achieved by endogenously expressing fluorescent proteins such as GFP, YFP, and mCherry, which emit light in the visible spectrum. However, many tissues in vivo also display high autofluorescence in this range (Tuchin, 2016; Zipfel et al., 2003), which can hinder the reliable detection of individual cancer cells through the centimeter-thick intact mouse body.

[0014] According to a preferred embodiment of the present invention, it is advantageous to label cells (e.g., cancer cells) using antibodies labeled with fluorescent dyes having emission peaks, particularly in the far-red range, in order to overcome such autofluorescence signals by providing a higher signal-to-background ratio, thereby reliably detecting single cells.

[0015] To achieve this goal, the inventors have developed an improved method for preparing animal tissues for fluorescence microscopy. Preferably, the method uses an antibody-based whole-body labeling (e.g., immunolabeling) technique to specifically label endogenous cellular proteins with fluorescent dyes such as Alexa and Atto dyes, preferably in the far-red spectrum, without relying on endogenously expressed fluorescent proteins. Organic solvent-based clearing methods, such as whole-body DISCO clearing methods (see Pan et al., 2016, which is incorporated herein by reference in its entirety for all purposes), can be included in the methods of the present invention. The advantages of the methods of the present invention are that they allow visualization of cells (e.g., cancer cells) in intact transparent mice even in tissues with high autofluorescence.

[0016] For example, the methods of the present invention can be used to assess tumor metastasis and the biodistribution of cancer cell targeting antibodies, for example in mice. For example, this finding can be exemplified by using mice transplanted with human breast cancer cells and injected with the therapeutic monoclonal antibody 6A10 against carbonic anhydrase XII (CA12) (for references to this antibody, see Battke et al., 2011; and Gondi et al., 2013, which are incorporated herein by reference in their entirety for all purposes). Thus, the present invention can advantageously detect spontaneous metastasis and monitor tumor drug-target interactions at the single cell level in intact mice, and further phenotype the defined tumor microenvironment by rehydration of transparent tissue and subsequent antibody labeling.

[0017] For example, the method can be used to analyze the distribution of micro-metastasis and therapeutic anti-tumor antibodies in tissues such as the whole body of mice at cellular resolution. The methods of the present invention are unbiased because they allow the labeling and detection of target molecules in animal tissues (e.g., whole mice) at single-cell resolution without the need to dissect the animal tissue before analyzing the animal tissue. Advantageously, according to the present invention, the animal tissue that can be prepared and analyzed at single-cell resolution without prior dissection is larger than previously known methods. Therefore, the deviation introduced by tissue dissection (and subsequent separate analysis of different anatomical parts of the animal tissue) can be minimized by the method of the present invention. For example, the deviation that may be introduced by analyzing only selected organs or parts of these organs can be minimized by the method of the present invention. In a non-limiting embodiment, the organic solvent used according to the method of the present invention helps to achieve this advantageous effect because it can shrink the animal tissue to a smaller size and make it easier for the animal tissue to be examined by fluorescence microscopy at a given maximum working distance through a microscope objective.

[0018] The methods of the present invention also have the advantage over previous methods that they can clear tissue including skin, for example, an entire adult mouse including skin.

[0019] The methods of the present invention also have the advantage that they can be easily applied to different laboratories without the need for highly specialized equipment, as even imaging using a common epifluorescence microscope can detect more details in intact transparent mice than can be observed by bioluminescence imaging.

[0020] For example, the methods of the present invention can also reduce the time and cost required to study tumor micrometastasis at the cellular level in the whole mouse body. In addition, because researchers can easily evaluate the entire mouse body instead of selected tissues / organs, and because of the high sensitivity of the method (capable of identifying and quantifying single cells in the whole body), the number of mice used for research can also be significantly reduced by the methods of the present invention. Therefore, the methods of the present invention proposed here can promote the translation of new therapies to the clinic more effectively than traditional methods.

[0021] In addition, unlike known tissue clearing methods (such as CUBIC and PACT methods) that make tissues brittle, the animal tissue preparation method for fluorescence microscopy according to the present invention can make animal tissues hard. Therefore, it is advantageous that the animal tissue obtained by the method of the present invention is suitable for dissection into different parts, and each part is further analyzed by fluorescence microscopy after dissection. It should be understood that according to the present invention, it is generally not necessary to dissect the animal tissue obtainable by the method of the present invention, because the animal tissue that can be prepared and analyzed at single cell resolution without prior dissection according to the present invention is larger than previously known methods. However, if dissection is required, animal tissue obtainable by the method of the present invention can be advantageously used. This is particularly useful for further characterizing micrometastases identified by the method of the present invention and their microenvironment after isolation.

[0022] Tissue labeling, such as whole-body immunostaining using antibodies

[0023] Imaging endogenous proteins (such as endogenous fluorescent proteins) in thick biological tissues faces significant challenges, including autofluorescence in the blue-green spectrum and photobleaching during long-term imaging and storage. In an exemplary embodiment of the present invention, in order to achieve high signal quality (e.g., for single tumor cell detection in a whole adult mouse), a first antibody (which binds to an endogenous protein, such as that of a cancer cell) can be labeled with a second antibody as a labeling agent, such as a second antibody conjugated to a fluorescent dye (e.g., Atto or Alexa dyes). The advantage of this approach is that it increases the signal-to-background ratio and allows visualization of single cells in tissue, especially even in mice that are several centimeters thick. According to the present invention, it should be understood that the use of fluorescent dyes in the far-red or longer wavelength spectrum, such as near-infrared fluorescent dyes, can be used to further improve imaging quality and may allow the study of subcellular structures / molecules in the whole mouse body (for examples of suitable fluorescent dyes, see Hong et al., 2017, which is incorporated herein by reference in its entirety for all purposes).

[0024] The present invention uses a fluorescent dye-containing labeling agent (e.g., an antibody conjugated to a fluorescent dye) for labeling, and the molecular weight of the labeling agent is preferably greater than 100 kDa, for example, equal to or greater than 110 kDa, equal to or greater than 120 kDa, equal to or greater than 130 kDa, or equal to or greater than 140 kDa.

[0025] Antibodies that can be conjugated with fluorescent dyes and used in the present invention include, but are not limited to, IgG molecules (eg, IgG1, IgG2, IgG3, or IgG4), IgD molecules, IgE molecules, IgA molecules, and IgM molecules.

[0026] The term "antibody" as used herein refers to any functional antibody that can specifically bind to a target antigen, as summarized in Chapter 7 of Paul, WE (Ed.): Fundamental Immunology 2nd Edition, Raven Press, Ltd., New York 1989, which is incorporated herein by reference. Without particular limitation, the term "antibody" encompasses antibodies from any appropriate source species, including chickens and mammals, such as mice, goats, non-human primates and humans. The antibody may be a monoclonal antibody or a polyclonal antibody. Such antibodies may be prepared by methods well known in the art. The term "antibody" also encompasses (without particular limitation) isolated antibodies and modified antibodies, such as genetically engineered antibodies, such as chimeric humanized or human antibodies. Preferred antibodies are shown in Tables 2 and 3.

[0027] In one embodiment, novel antibodies can be generated for use in the methods and uses of the invention to study pathologies that affect the entire body. For example, markers (e.g., antibodies) that can be used as markers of inflammation or infection facilitate collecting unbiased readouts of inflammatory diseases (e.g., multiple sclerosis or rheumatoid arthritis) in whole mice or infectious diseases that affect the entire body.

[0028] Detection of micrometastasis according to the present invention

[0029] Unbiased, high-throughput mapping of tumor micrometastases at cellular resolution (e.g., in whole rodents) can be a useful tool to reveal the biological principles behind tumor cell dissemination. In an exemplary embodiment, the present invention encompasses a wildDISCO method that can be used for volumetric imaging of tumor micrometastases throughout the body of a mouse. Although the use of a single-plane laser scanning light sheet microscope is the most preferred embodiment of the analysis method according to the present invention, such as detecting cancer cells in transparent mice, new insights can be provided even using a standard fluorescence microscope. In addition, epifluorescence imaging facilitates direct scanning of the transparent mouse body within a few minutes to identify the target area before collecting a large data set with a light sheet microscope. Subsequent light sheet microscopy imaging can focus only on the target organ / area based on the epifluorescence data. This approach will greatly speed up the research conducted and reduce the amount of data that needs to be analyzed.

[0030] Advantageously, the methods of the invention can be adapted to detect and map cancer metastasis throughout the mouse body at the cellular level, thereby identifying the precise location of individual disseminated cancer cells. The methods of the invention allow re-probing of identified metastatic tissues with conventional antibodies, gene expression analysis by, for example, RNAseq and proteomics (by mass spectrometry).

[0031] Therefore, according to the present invention, the method for preparing animal tissue for fluorescence microscopy of the present invention is advantageous in that it preserves proteins (functional epitopes) and DNA / RNA.

[0032] Thus, the methods of the present invention allow for further characterization and molecular screening of micrometastases and single tumor cells found in distant organs. According to the present invention, the use of molecular markers for specific subtypes of tumor cells (e.g., cancer stem cells) or inflammatory cells and extracellular matrix components (e.g., cancer-associated fibroblasts, T cells, and macrophages) in the tumor microenvironment will help determine their precise spatiotemporal distribution in tissues (e.g., the entire rodent body) during metastasis.

[0033] Biodistribution analysis of biopharmaceutical drugs according to the present invention

[0034] While accurate assessment of the biodistribution of biopharmaceuticals, such as antibody drugs, is critical to evaluating their specificity and utility for treatments such as tumor therapy, there is currently no method that can provide such information at the cellular level in intact organisms. The method of the present invention (also referred to as the "wildDISCO" method in an exemplary embodiment) is a new tool that can be used not only to study the distribution of individual tumor cells, but also to study the distribution of antibody-based therapeutics. The method of the present invention can identify tumor cells targeted by antibodies, particularly metastases in different organs, including the lungs, kidneys, brain, and liver. The analytical methods of the present invention are also advantageous in that they can also be used to detect the binding of biopharmaceuticals, such as therapeutic antibodies, to non-target tissues (e.g., non-cancerous tissues in the case of cancer therapeutic antibodies) to indicate potential off-target effects.

[0035] Biodistribution analysis of the nanoparticles according to the invention

[0036] In an exemplary method for analyzing the biodistribution of nanoparticles, nanoparticles (DNA origami or carbon nanotubes) can be conjugated to polymers (e.g., PEG) to increase circulation time and stability. They can also be labeled with moieties such as antibodies, peptides, aptamers to achieve targeting. For example, they can be targeted to immune cells using CpG peptides. Finally, they can also be conjugated with fluorescent dyes (e.g., Alexa or Atto dyes) used in accordance with the methods of the invention. The conjugated nanoparticles can be dissolved in PBS at a concentration of 200 nM-2µM. 100-200µL of this solution is then injected intravenously or intraperitoneally into mice. Subsequently, the mice are perfused as early as 3 hours (or longer). The biodistribution of the nanoparticles is evaluated by the methods of the invention.

[0037] Thus, the present invention provides an advantageous labeling and analysis platform. For example, the platform can visualize and analyze tumor micrometastases and antibody-based therapies in whole mice at single-cell resolution. Because the methods of the present invention save time and cost, they can be used to study a variety of biomedical problems, such as those related to various pathological or developmental processes that affect the entire organism.

[0038] Therefore, the present invention encompasses the following preferred embodiments:

[0039] Implementation

[0040] 1. A method for preparing animal tissue for fluorescence microscopy, the method comprising the following steps:

[0041] a) optionally decalcifying the fixed animal tissue using a decalcifying solution;

[0042] b) optionally decolorizing the fixed animal tissue with a heme-removing solution;

[0043] c) labeling a target molecule in a fixed animal tissue with a labeling solution, wherein the labeling solution comprises a labeling agent containing a fluorescent dye capable of binding to the target molecule, wherein the labeling agent preferably has a molecular weight greater than 100 kDa, so as to obtain a fixed animal tissue labeled with the labeling agent containing a fluorescent dye,

[0044] wherein the fixed animal tissue is treated with a permeabilization solution, preferably before and / or during the labeling of the target molecule in step c), and

[0045] Preferably, wherein the permeabilization solution and the labeling solution are the same or different solutions,

[0046] and

[0047] wherein the permeabilization solution and / or the labeling solution comprises a cyclodextrin derivative; and

[0048] d) Optionally, clearing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent using a clearing solution containing an organic solvent; thereby obtaining the animal tissue for fluorescence microscopy.

[0049] 2. The method according to embodiment 1, wherein the cyclodextrin derivative has a structure shown in the following formula:

[0050]

[0051] in:

[0052] m is 6 to 8;

[0053] R 2 , R 3and R 6 are each independently selected from H and optionally substituted alkyl; and

[0054] The degree of substitution (DS) is the number of non-hydrogen groups R per pyranose unit. 2 , R 3 and R 6 The average number is 0 to 3.

[0055] 3. A method according to embodiment 2, wherein the optionally substituted alkyl group is a straight or branched C1-C6 alkyl group optionally substituted with one or more groups selected from OH, SO3H, SO3Na, oxo and COOH.

[0056] 4. The method according to any one of embodiments 2 and 3, wherein R 2 , R 3 and R 6 Each is independently selected from H and a linear or branched C1-C4 alkyl group optionally substituted with one or more groups selected from OH, SO3H, SO3Na, oxo and COOH.

[0057] 5. The method according to any one of embodiments 2 to 4, wherein DS ≥ 0.4.

[0058] 6. The method according to any one of embodiments 2 to 5, wherein DS ≥ 0.5.

[0059] 7. The method according to any one of embodiments 2 to 6, wherein DS ≥ 0.6.

[0060] 8. The method according to any one of embodiments 2 to 7, wherein DS ≥ 0.7.

[0061] 9. The method according to any one of embodiments 2 to 8, wherein DS ≥ 0.8.

[0062] 10. The method according to any one of embodiments 2 to 9, wherein DS ≥ 0.9.

[0063] 11. The method according to any one of embodiments 2 to 10, wherein DS ≥ 1.0.

[0064] 12. The method according to any one of embodiments 2 to 11, wherein DS ≥ 1.5.

[0065] 13. The method according to any one of embodiments 2 to 12, wherein DS ≥ 1.8.

[0066] 14. The method according to any one of embodiments 2 to 13, wherein DS ≥ 1.9.

[0067] 15. The method according to any one of embodiments 2 to 14, wherein DS ≥ 2.0.

[0068] 16. The method according to any one of embodiments 2 to 15, wherein DS ≥ 2.5.

[0069] 17. The method according to any one of embodiments 2 to 16, wherein DS ≤ 3.0.

[0070] 18. The method according to any one of embodiments 2 to 17, wherein DS ≤ 2.8.

[0071] 19. The method according to any one of embodiments 2 to 18, wherein R 2 , R 3 and R 6 Each is independently selected from H and a linear or branched C1-C6 alkyl group optionally substituted with one or more oxo and / or OH.

[0072] 20. The use according to any one of the preceding embodiments, wherein the cyclodextrin derivative is not methyl-β-cyclodextrin with a degree of substitution of 1.8.

[0073] 21. The use according to any one of the preceding embodiments, wherein the cyclodextrin derivative is not methyl-β-cyclodextrin with a degree of substitution <2.0.

[0074] 22. The method according to any one of embodiments 2 to 21, provided that when R 2 , R 3 and R 6 When each is selected from H and CH3, then DS≥2.

[0075] 23. The method according to any one of embodiments 2 to 22, wherein DS is from 1.8 to 2.0.

[0076] 24. The method according to any one of embodiments 2 to 23, wherein R 2 , R 3 and R 6 Each is independently selected from H and CH3.

[0077] 25. The method according to any one of embodiments 2 to 24, wherein R 2 , R 3 and R 6 Each is independently selected from H and CH3.

[0078] 26. The method of embodiment 25, wherein the DS is 1.8.

[0079] 27. The method according to any one of embodiments 2 to 23, wherein

[0080] (a) R 2 and R 6 is a linear or branched C1-C6 alkyl group, and R 3 is H; or

[0081] (b) R 2 and R 3 is a linear or branched C1-C6 alkyl group, and R 6 is H; or

[0082] (c) R 3 and R 6 is a linear or branched C1-C6 alkyl group, and R 2 It's H.

[0083] 28. The method according to any one of embodiments 2 to 26, wherein R 2 and R 6 is CH3, and R 3 It's H.

[0084] 29. The method according to any one of embodiments 2 to 26, wherein R 2 , R 3 and R 6 Each independently selected from H and C(O)C 1-5 alkyl.

[0085] 30. The method according to embodiment 29, wherein R 2 , R 3 and R 6 Each is independently selected from H and C(O)CH3.

[0086] 31. The method according to any one of embodiments 2 to 30, wherein DS is from 2.5 to 3.0.

[0087] 32. The method according to any one of embodiments 2 to 31, wherein R 2 , R 3 and R 6 Each is C(O)CH3.

[0088] 33. The method according to any one of embodiments 2 to 23, wherein R 2 , R 3 and R 6 Each independently selected from H, -C 1-2 Alkyl (OH) C 1-3 Alkyl and -C 1-2 Alkyl-OH.

[0089] 34. The process according to any one of embodiments 2 to 33, provided that when the alkyl group is substituted with one or more OH groups, then the alkyl group is a linear or branched C3-C6 alkyl group and / or DS ≥ 0.9.

[0090] 35. The method of any one of embodiments 2 to 34, wherein DS is from 0.8 to 3.0.

[0091] 36. The method of any one of embodiments 2 to 35, wherein DS is from 0.9 to 3.0.

[0092] 37. The method according to any one of embodiments 2 to 36, wherein R 2 , R 3 and R 6 Each is independently selected from H and -CH2CH(OH)CH3.

[0093] 38. The method of any one of embodiments 2 to 37, wherein DS is 0.9.

[0094] 39. The method according to any one of embodiments 2 to 38, wherein R 2 , R 3 and R 6 Each is independently selected from H and -CH2CH2OH.

[0095] 40. The method of embodiment 39, wherein DS is 0.8.

[0096] 41. The method according to any one of embodiments 2 and 40, wherein R 2 , R 3 and R 6 Each is independently selected from H and a linear or branched C1-C5 alkyl group optionally substituted with COOH.

[0097] 42. The method according to any one of embodiments 2 to 41, wherein R 2 , R 3 and R 6 are each independently selected from H and –C(O)–C 1-3 Alkyl –COOH.

[0098] 43. The method according to any one of embodiments 2 to 42, wherein R 2 , R 3 and R 6 Each is independently selected from H and -C(O)CH2CH2COOH.

[0099] 44. The method of any one of embodiments 41 to 43, wherein DS is from 0.4 to 0.7.

[0100] 45. The method of any one of embodiments 2 to 44, wherein DS is 0.5.

[0101] 46. ​​The method according to any one of embodiments 2 to 45, wherein m is 6 or 8.

[0102] 47. The method according to any one of embodiments 2 to 46, wherein m is 7.

[0103] 48. A method according to any one of the preceding embodiments, wherein the cyclodextrin derivative is selected from (2-hydroxypropyl)-β-cyclodextrin, triacetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, heptath(2,6-di-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, γ-cyclodextrin and α-cyclodextrin; more preferably (2-hydroxypropyl)-β-cyclodextrin or heptath(2,6-di-O-methyl)-β-cyclodextrin; most preferably heptath(2,6-di-O-methyl)-β-cyclodextrin.

[0104] 49. The method according to any one of the preceding embodiments, further comprising a blocking step for blocking non-specific antigen binding of the antibody, wherein the blocking step is performed by treating the fixed animal tissue with a blocking solution prior to the labeling step.

[0105] 50. A method according to embodiment 49, wherein the blocking solution comprises animal serum.

[0106] 51. The method according to embodiment 50, wherein the animal serum is mammalian serum, preferably goat serum or donkey serum, more preferably goat serum.

[0107] 52. The method of any one of embodiments 49 to 51, wherein the locking solution further comprises a surfactant.

[0108] 53. The method according to embodiment 53, wherein the surfactant is a nonionic surfactant, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100.

[0109] 54. The method of any one of embodiments 49 to 53, wherein the locking solution comprises:

[0110] In a buffered aqueous solution, preferably phosphate buffered saline (PBS),

[0111] - animal serum at a concentration of 1 to 15, preferably 3 to 10, more preferably 3% v / v; and / or

[0112] - a nonionic surfactant in a concentration of 0.5 to 4, preferably 1 to 3, more preferably 2% w / v,

[0113] The buffered aqueous solution, preferably phosphate buffered saline, optionally has a buffer concentration of 0.05 to 0.2M, preferably 0.08 to 1.2M, more preferably 0.1M.

[0114] 55. The method according to any one of the preceding embodiments, wherein treatment with a permeabilization solution is performed simultaneously with treatment with a blocking solution.

[0115] 56. The method according to any one of the preceding embodiments, wherein the permeabilization solution and the blocking solution are the same solution.

[0116] 57. The method according to any one of the preceding embodiments, wherein the method comprises step a).

[0117] 58. The method according to any one of the preceding embodiments, wherein in step a), the decalcification solution is selected from a solution comprising EDTA and NaHCO 3 , a solution comprising formic acid, a solution comprising HNO 3 , or a solution comprising HCl;

[0118] Preferably, the pH value of the decalcification solution is 8-9.

[0119] 59. The method according to any one of the preceding embodiments, wherein the fixed animal tissue is obtainable by fixation with a fixative solution comprising paraformaldehyde, optionally 4±2% w / v paraformaldehyde and optionally heparin.

[0120] 60. The method according to any one of embodiments 57 to 59, wherein a blocking step as defined in any one of embodiments 49 to 56 is performed after step a).

[0121] 61. The method according to any one of the preceding embodiments, wherein the method comprises step b).

[0122] 62. The method according to any one of the preceding embodiments, wherein step b) is performed by perfusing the fixed animal tissue with the heme-depleted solution, and / or wherein the heme-depleted solution is a heme chelating solution.

[0123] 63. The method according to any one of the preceding embodiments, wherein in step b), the heme-removing solution comprises an amino alcohol suitable for removing heme and optionally a surfactant.

[0124] 64. A method according to embodiment 63, wherein the heme-removing solution contains a surfactant, and wherein the surfactant is an ionic surfactant, a non-ionic surfactant, a zwitterionic surfactant, a chaotropic surfactant, or a combination thereof.

[0125] 65. The method of embodiment 64, wherein the surfactant is an ionic surfactant which is sodium dodecyl sulfate or sodium deoxycholate.

[0126] 66. A method according to embodiment 64, wherein the surfactant is a non-ionic surfactant, which is 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, tert-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether or polyoxyethylene (20) sorbitan monolaurate.

[0127] 67. The method of embodiment 64, wherein the surfactant is a zwitterionic surfactant which is 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate hydrate.

[0128] 68. The method of embodiment 64, wherein the surfactant is a chaotropic surfactant which is urea.

[0129] 69. The method of any one of embodiments 63-68, wherein the amino alcohol is N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, N-butylethanolamine, N-methyldiethanolamine, 4-(2-hydroxyethyl)morpholine, N-ethyldiethanolamine, 2-(diisopropylamino)ethanol, 4-methylmorpholine N-oxide, or 1-(2-hydroxyethyl)piperidine.

[0130] 70. The method of any one of embodiments 63 to 69, wherein the amino alcohol is N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine.

[0131] 71. A method according to embodiment 70, wherein the heme-removing solution is a 1:2 or 1:3 dilution of the following reagents, preferably in 0.1M PBS: 25wt% urea, 25wt% N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, 15wt% Triton X-100 in 0.1M PBS.

[0132] 72. The method according to any one of the preceding embodiments, wherein in step b), the heme-removing solution comprises an oxidizing agent for oxidizing heme.

[0133] 73. The method of embodiment 72, wherein the oxidizing agent for oxidizing heme is benzoyl peroxide, 3-chloroperbenzoic acid, or magnesium monoperoxyphthalate hexahydrate.

[0134] 74. A method according to embodiment 72, wherein the oxidizing agent used to oxidize heme is benzoyl peroxide.

[0135] 75. The method according to any one of the preceding embodiments, wherein step b) is performed simultaneously with step c).

[0136] 76. The method according to any one of the preceding embodiments, wherein the solution for removing heme is the same solution as the permeabilization solution and / or the labeling solution.

[0137] 77. A method according to any of the preceding embodiments, wherein the permeabilization solution and / or the labeling solution comprises a surfactant, and wherein the surfactant is an ionic surfactant, a non-ionic surfactant, a zwitterionic surfactant, a chaotropic surfactant, or a combination thereof.

[0138] 78. The method according to any one of the preceding embodiments, wherein the permeabilization solution and / or the labeling solution comprises a non-ionic surfactant and / or a zwitterionic surfactant.

[0139] 79. The method according to any one of the preceding embodiments, wherein the permeabilization solution and / or the labeling solution comprises:

[0140] A nonionic surfactant, which is preferably selected from Triton X-100 and IGEPAL CA-630, more preferably Triton X-100; and

[0141] Optionally a zwitterionic surfactant selected from CHAPS or CHAPSO, more preferably CHAPS.

[0142] 80. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is equal to or greater than 110 kDa.

[0143] 81. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is equal to or greater than 120 kDa.

[0144] 82. A method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is equal to or greater than 130 kDa.

[0145] 83. A method according to any of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is equal to or greater than 140 kDa.

[0146] 84. A method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is equal to or greater than 150 kDa.

[0147] 85. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 900 kDa or less.

[0148] 86. A method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 500 kDa or less.

[0149] 87. A method according to any of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 385 kDa or less.

[0150] 88. A method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 300 kDa or less.

[0151] 89. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 200 kDa or less.

[0152] 90. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 180 kDa or less.

[0153] 91. A method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150 kDa or less.

[0154] 92. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 110 to 900 kDa.

[0155] 93. A method according to any of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 120 to 500 kDa.

[0156] 94. A method according to any of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 130 to 385 kDa.

[0157] 95. The method according to any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 140 to 300 kDa.

[0158] 96. A method according to any of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150 to 200 kDa.

[0159] 97. A method according to any of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150 to 180 kDa.

[0160] 98. A method according to any one of the preceding embodiments, wherein the fluorescent dye is capable of emitting infrared or red fluorescence.

[0161] 99. The method according to any one of the preceding embodiments, wherein the fluorescent dye is capable of emitting near-infrared or far-red fluorescence.

[0162] 100. The method according to any one of the preceding embodiments, wherein the emission maximum of the fluorescent dye is at a wavelength above 480 nm.

[0163] 101. A method according to any one of embodiments 1 to 100, wherein the emission maximum of the fluorescent dye is at a wavelength above 500 nm.

[0164] 102. A method according to any one of embodiments 1 to 101, wherein the emission maximum of the fluorescent dye is at a wavelength above 550 nm.

[0165] 103. A method according to any one of embodiments 1 to 102, wherein the emission maximum of the fluorescent dye is at a wavelength higher than 590 nm.

[0166] 104. A method according to any one of embodiments 1 to 103, wherein the emission maximum of the fluorescent dye is at a wavelength above 600 nm.

[0167] 105. A method according to any one of embodiments 1 to 104, wherein the emission maximum of the fluorescent dye is at a wavelength higher than 640 nm.

[0168] 106. A method according to any one of embodiments 1 to 105, wherein the emission maximum of the fluorescent dye is at a wavelength above 700 nm.

[0169] 107. A method according to any one of embodiments 1 to 106, wherein the emission maximum of the fluorescent dye is in the wavelength range of 640nm to 700nm.

[0170] 108. A method according to any one of embodiments 1 to 107, wherein the emission maximum of the fluorescent dye is at a wavelength below 1000 nm or below 900 nm.

[0171] 109. A method according to any one of embodiments 1 to 108, wherein the emission maximum of the fluorescent dye is at a wavelength below 800 nm.

[0172] 110. A method according to any one of the preceding embodiments, wherein the fluorescent dye-containing labeling agent is an antibody conjugated to the fluorescent dye, the antibody being capable of binding to the target molecule; and is preferably selected from the antibodies listed in Table 2 and Table 3.

[0173] 111. The method of embodiment 110, wherein the antibody is IgG, IgA, IgM, IgD or IgE.

[0174] 111. The method of embodiment 110 or 111, wherein the antibody is IgG.

[0175] 112. The method of embodiment 111, wherein the antibody is IgG1.

[0176] 113. A method according to embodiment 111, wherein the antibody is IgG2.

[0177] 114. The method of embodiment 111, wherein the antibody is IgG3.

[0178] 115. The method of embodiment 111, wherein the antibody is IgG4.

[0179] 116. The method according to any one of the preceding embodiments, wherein step c) is performed by perfusing the fixed animal tissue with a labeling solution comprising the labeling agent comprising the fluorescent dye.

[0180] 117. The method according to any one of the preceding embodiments, wherein the fluorescent dye-containing labeling agent is a fluorescent dye that is capable of binding to the target molecule.

[0181] 118. A method according to any one of the preceding embodiments, wherein the fluorescent dye-containing marking agent comprises a fluorescent dye, preferably, wherein the fluorescent dye is Nissl, propidium iodide, methoxy-x04, acetocresyl violet, Pyronin Y, thiazine red, lectin, DiI, Atto dye, Alexa Fluor dye, Cy dye and To-pro3.

[0182] 119. A method according to any of the preceding embodiments, wherein the fluorescent dye-containing labeling agent comprises a fluorescent dye selected from Alexa Fluor 568, Alexa Fluor 647, Alexa Fluor 750, Atto 550, Atto 647, Cy7, Cy5 and Cy3.

[0183] 120. The method according to any one of the preceding embodiments, wherein the refractive index of the organic solvent deviates from the refractive index of the animal tissue by no more than 5%.

[0184] 121. A method according to any one of the preceding embodiments, wherein the refractive index of the clearing agent comprising an organic solvent deviates from the refractive index of the animal tissue by no more than 2%.

[0185] 122. The method according to any one of the preceding embodiments, wherein the clearing solution comprising an organic solvent has a refractive index between 1.500 and 1.600.

[0186] 123. The method according to any of the preceding embodiments, wherein the clearing solution comprising an organic solvent has a refractive index between 1.520 and 1.580.

[0187] 124. The method according to any one of the preceding embodiments, wherein the organic solvent comprises benzyl alcohol, benzyl benzoate, dibenzyl ether, 3-phenyl-2-acrylate ethyl ester, 3-phenylacrylate allyl ester, PEG (Mn=200-1000), PEGDA (Mn=200-1000), PEGMA (Mn=200-1000), 1-phenylnaphthalene and / or diphenyl ether.

[0188] 125. The method according to any one of the preceding embodiments, wherein the clearing solution comprising an organic solvent further comprises an antioxidant.

[0189] 126. The method according to any one of the preceding embodiments, wherein the clearing solution comprising an organic solvent consists of benzyl alcohol, benzyl benzoate and diphenyl ether in a volume ratio of 4:8:3 to 10:20:3 and the antioxidant.

[0190] 127. The method of embodiment 125 or 126, wherein the antioxidant is DL-alpha tocopherol.

[0191] 128. The method according to embodiment 125 or 126 or 127, wherein the antioxidant is present in the clearing solution in an amount of 0.4 vol%.

[0192] 129. The method according to any one of the preceding embodiments, wherein step d) is performed by perfusing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent with the clearing solution comprising an organic solvent.

[0193] 130. A method according to embodiment 129, wherein the fixed animal tissue labeled with the fluorescent dye-containing labeling agent is perfused with the clearing solution containing an organic solvent for at least 6 hours.

[0194] 131. The method according to embodiment 129 or 130, wherein step d) further comprises, prior to perfusing with the clearing solution, perfusing with a dehydrating solution with an increasing gradient comprising 0 vol% to 100 vol% of an additional organic solvent.

[0195] 132. A method according to embodiment 131, wherein perfusion with a dehydration solution comprising an increasing gradient of 0 vol% to 100 vol% of the additional organic solvent is followed by perfusion with a degreasing solution comprising another organic solvent.

[0196] 133. A method according to embodiment 132, wherein the additional organic solvent is tert-butanol, tetrahydrofuran (THF), methanol, ethanol or 1,4-dioxane, and wherein the perfusion performed with the gradient increase is performed at a temperature above the melting temperature of the additional organic solvent.

[0197] 134. A method according to embodiment 132 or 133, wherein the another organic solvent is dichloromethane, chloroform, methanol, hexane, butanol, ethyl acetate, tert-butyl methyl ether, and wherein the infusion with the another organic solvent is performed at a temperature above the melting temperature of the another organic solvent.

[0198] 135. The method according to any one of the preceding embodiments, wherein the marking solution and the permeabilizing solution and the clearing solution are actively delivered by applying pressure, preferably by applying pressure with a pump.

[0199] 136. The method according to any one of the preceding embodiments, wherein said labeling of target molecules with a labeling solution and said treating with a permeabilization solution are performed by perfusion at a pressure higher than 80 mmHg, preferably higher than 150 mmHg.

[0200] 137. The method according to any one of the preceding embodiments, wherein said labeling of target molecules with a labeling solution and said treating with a permeabilization solution are performed by perfusion at a pressure of 220 to 240 mmHg, preferably above 230 mmHg.

[0201] 138. A method according to any one of the preceding embodiments, wherein the fixed and decolorized animal tissue is treated with a permeabilization solution prior to the labeling of target molecules in step c), and wherein the permeabilization solution and the labeling solution are different solutions.

[0202] 139. A method according to embodiment 138, wherein the permeabilization solution is a dehydration solution as defined in any of embodiments 132 or 133.

[0203] 140. The method according to embodiment 138, wherein the permeabilization solution is a defatting solution as defined in any one of embodiments 132 or 133.

[0204] 141. The method of embodiment 138, wherein the permeabilization solution comprises acetic acid.

[0205] 142. The method of embodiment 138, wherein the permeabilization solution comprises guanidine hydrochloride and / or sodium acetate.

[0206] 143. A method according to any one of the preceding embodiments 1 to 137, wherein the fixed animal tissue is treated with a permeabilization solution during the labeling of target molecules in step c), and wherein the permeabilization solution and the labeling solution are the same solution.

[0207] 144. The method according to any of the preceding embodiments, wherein step b) is performed by perfusing said fixed animal tissue with said heme-depleted solution at a pressure higher than 80 mmHg, preferably higher than 150 mmHg.

[0208] 145. The method according to any of the preceding embodiments, wherein step b) is performed by perfusing said fixed animal tissue with said heme-depleted solution at a pressure of 220 to 240 mmHg, preferably above 230 mmHg.

[0209] 146. The method according to any one of embodiments 130 to 145, wherein the perfusion in step d) is performed at a pressure higher than 80 mmHg, preferably higher than 150 mmHg.

[0210] 147. The method according to any one of embodiments 130 to 145, wherein the perfusion in step d) is performed at a pressure of 220 to 240 mmHg, preferably at a pressure above 230 mmHg.

[0211] 148. The method according to any one of the preceding embodiments, wherein the permeabilization solution comprises a cyclodextrin derivative as defined in any one of embodiments 2 to 48.

[0212] 149. The method according to any one of the preceding embodiments, wherein the marking solution comprises a cyclodextrin derivative as defined in any one of embodiments 2 to 48.

[0213] 150. The method according to any one of the preceding embodiments, wherein the marking solution is a composition as defined in any one of aspects 233 to 242 comprising a marking agent comprising a fluorescent dye.

[0214] 151. The method of any one of the preceding embodiments, wherein the animal tissue is from a mammal.

[0215] 152. A method according to any of the preceding embodiments, wherein the animal tissue is from a non-human mammal or a human.

[0216] 153. A method according to any of the preceding embodiments, wherein the animal tissue is from a rodent.

[0217] 154. A method according to any of the preceding embodiments, wherein the animal tissue is from a mouse.

[0218] 155. A method according to any of the preceding embodiments, wherein the animal tissue is a whole mouse.

[0219] 156. A method according to any of the preceding embodiments, wherein the animal tissue is pig brain.

[0220] 157. The method of any one of the preceding embodiments, wherein the animal tissue is a whole organ or a portion thereof.

[0221] 158. The method according to any one of the preceding embodiments, wherein the target molecule labeled by the labeling agent in step c) is:

[0222] (i) a structure present in the fixed animal tissue, preferably a protein, a lipid, a DNA or an RNA, more preferably a protein present in the fixed animal tissue; or

[0223] (Ii) a first antibody that binds to a structure present in the fixed animal tissue, preferably a protein, a lipid, a DNA or an RNA, more preferably a protein present in the fixed animal tissue.

[0224] 159. A method according to any one of the preceding embodiments, wherein the animal tissue contains cancer, and wherein the target molecule labeled with the marker in step c) is a structure present in the cancer, preferably a protein, lipid, DNA or RNA, more preferably a protein present in the cancer.

[0225] 160. A method according to any one of the preceding embodiments, wherein the animal tissue contains cancer metastasis, and wherein the target molecule labeled with the marker in step c) is a structure present in the cancer, preferably a protein, lipid, DNA or RNA, more preferably a protein present in the cancer.

[0226] 161. A method according to any of the preceding embodiments, wherein the animal has been treated with a biopharmaceutical drug, wherein the animal tissue contains the biopharmaceutical drug, and wherein the biopharmaceutical drug is the target molecule labeled by the labeling agent in step c), or wherein the biopharmaceutical drug has been labeled with an additional fluorescent dye in vitro, or wherein the biopharmaceutical drug itself is fluorescent.

[0227] 162. The method of embodiment 161, wherein the biopharmaceutical drug is a small molecule.

[0228] 163. The method of embodiment 161, wherein the biopharmaceutical drug is a therapeutic protein.

[0229] 164. The method of embodiment 161, wherein the biopharmaceutical drug is a therapeutic antibody.

[0230] 165. A method according to any of the preceding embodiments, wherein the method is not a method of treatment of the human or animal body by surgery or therapy, nor is it a diagnostic method performed on the human or animal body.

[0231] 166. A method according to any of the preceding embodiments, wherein the method is an ex vivo method.

[0232] 167. A method according to any of the preceding embodiments, wherein the volume of animal tissue obtained in step d) for fluorescence microscopy is smaller than the volume of the fixed animal tissue used in step b).

[0233] 168. A method according to embodiment 167, wherein the volume of animal tissue obtained in step d) for fluorescence microscopy is 40% to 75% smaller than the volume of the fixed animal tissue used in step b).

[0234] 169. The method according to any one of the preceding embodiments, further comprising the following steps before labeling the target molecule in the fixed animal tissue with the labeling solution:

[0235] (Ii) contacting the fixed animal tissue with a first antibody capable of binding to a structure present in the fixed animal tissue, preferably a protein, a lipid, a DNA or an RNA, more preferably a protein present in the fixed animal tissue,

[0236] The labeling agent containing a fluorescent dye can bind to the first antibody.

[0237] 170. The method according to embodiment 169, wherein step c.1) is performed by perfusing the fixed animal tissue with a solution comprising the first antibody.

[0238] 171. The method according to embodiment 169 or 170, wherein step c.1) is performed by perfusing the fixed animal tissue with a first labeling solution comprising the first antibody and the cyclodextrin derivative as defined in any one of embodiments 2 to 48.

[0239] 172. A method according to any one of embodiments 169 to 171, wherein the first antibody is not conjugated to a fluorescent dye; and is preferably selected from those listed in Table 2 and Table 3.

[0240] 173. The method of any one of embodiments 169 to 172, wherein the first antibody is present in a composition as defined in any one of embodiments 233 to 242.

[0241] 174. The method of any one of embodiments 169 to 173, wherein the first antibody has a molecular weight as defined in any one of embodiments 80 to 97.

[0242] 175. The method of any one of embodiments 169 to 174, wherein the first antibody w is IgG, IgA, IgM, IgD or IgE.

[0243] 176. The method of any one of embodiments 169 to 175, wherein the first antibody is IgG.

[0244] 177. A method according to embodiment 176, wherein the first antibody is IgG1, IgG2, IgG3 or IgG4.

[0245] 178. The method of any one of embodiments 169 to 177, wherein the first antibody is a rabbit antibody or a rat antibody.

[0246] 179. The method according to any one of the preceding embodiments, comprising the following steps, preferably in this order:

[0247] - destaining, permeabilization and blocking steps, which are performed by treating the fixed animal tissue with heme-depleting, permeabilizing and blocking solutions;

[0248] - labeling the target molecule in the fixed animal tissue with a labeling solution, wherein the labeling solution comprises a cyclodextrin derivative and a labeling agent containing a fluorescent dye capable of binding to the target molecule, wherein the labeling agent preferably has a molecular weight greater than 100 kDa, so as to obtain the fixed animal tissue labeled with the labeling agent containing the fluorescent dye.

[0249] 180. The method according to any one of the preceding embodiments, comprising the following steps, preferably in this order:

[0250] - Destaining, permeabilization and blocking steps of fixed animal tissues by treatment with heme-removing, permeabilization and blocking solutions;

[0251] - contacting the fixed animal tissue with a first labeling solution comprising a cyclodextrin derivative and a first antibody capable of binding to a structure present in the fixed animal tissue, preferably a protein, a lipid, a DNA or an RNA, more preferably a protein present in the fixed animal tissue; and

[0252] - Labeling the first antibody that has bound to the structure in the fixed animal tissue with a second labeling solution comprising a cyclodextrin derivative and a labeling agent containing a fluorescent dye that can bind to the first antibody, wherein the labeling agent preferably has a molecular weight greater than 100 kDa, so as to obtain a fixed animal tissue labeled with the labeling agent containing a fluorescent dye.

[0253] 181. The method of embodiment 179 or 180, wherein the heme removal, permeabilization, and blocking solutions are the same solution ("pretreatment solution").

[0254] 182. A method according to embodiment 181, wherein the permeabilization solution has the composition of the blocking solution as defined in any one of aspects 49 to 55.

[0255] 183. A method according to any one of embodiments 169 to 182, wherein the first marking solution has a composition as defined in any one of aspects 233 to 242 and comprises a first antibody.

[0256] 184. A method according to any one of embodiments 169 to 183, wherein labeling with a labeling agent comprising a fluorescent dye is performed using a second labeling solution having a composition as defined in any one of aspects 233 to 242 and comprising a labeling agent comprising a fluorescent dye.

[0257] 185. The method of any one of embodiments 179 to 184, further comprising decalcifying the fixed animal tissue with a decalcifying solution prior to the destaining, permeabilizing, and blocking steps.

[0258] 186. The method of any one of embodiments 179 to 185, further comprising clearing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent with a clearing solution comprising an organic solvent after one or more labeling steps.

[0259] 187. Animal tissue or animal body obtainable by the method for preparing animal tissue for fluorescence microscopy according to any one of the preceding embodiments, wherein said animal tissue contains said target molecule labeled with said fluorescent dye-containing labeling agent.

[0260] 188. The animal tissue of embodiment 187, wherein the animal tissue is a whole rodent, preferably a whole mouse.

[0261] 189. The animal tissue of embodiment 187, wherein the animal tissue is a whole organ or a portion thereof.

[0262] 190. The animal tissue of embodiment 189, wherein the animal tissue is a whole mammalian organ.

[0263] 191. The animal tissue of any one of embodiments 187 to 190, having a size of at least 1 mm long × at least 1 mm wide × at least 1 mm high; preferably at least 5 mm long × at least 5 mm wide × at least 5 mm high, more preferably at least 1 cm long × at least 1 cm wide × at least 1 cm high.

[0264] 192. The animal tissue of any one of embodiments 187 to 191, wherein the animal tissue is a tissue block having a size of 2×2×2 cm.

[0265] 193. An animal tissue according to any of the preceding embodiments, wherein in the animal tissue, the target molecules are labeled with the fluorescent dye-containing marker, and wherein when analyzed by fluorescence microscopy, preferably light sheet fluorescence microscopy, all target molecules can be detected at single-cell resolution regardless of their location in the animal tissue.

[0266] 194. A method for analyzing an animal tissue or an animal body according to any one of embodiments 187 to 193, the method comprising step i) analyzing the tissue by fluorescence microscopy to detect the fluorescence of the fluorescent dye in the animal tissue.

[0267] 195. An analytical method according to embodiment 194, wherein the method further comprises step ii) visualizing the detected fluorescence of the fluorescent dye to obtain an image of the animal tissue, preferably a three-dimensional image.

[0268] 196. An analytical method according to embodiment 195, wherein the image is an image with single-cell resolution throughout the animal tissue.

[0269] 197. An analysis method according to any of the preceding embodiments, wherein the thickness of the animal tissue does not exceed 20 cm, preferably does not exceed 10 cm, and more preferably does not exceed 5 cm.

[0270] 198. The analytical method according to any of the preceding embodiments, wherein the thickness of the animal tissue does not exceed 2 cm, preferably is 1.5 to 2 cm thick.

[0271] 199. The analysis method according to any one of the preceding embodiments, wherein the analysis method further comprises a method according to any one of embodiments 1-186 before step i).

[0272] 200. The analytical method according to any of the preceding embodiments, wherein the fluorescence microscopy is selected from the group consisting of light sheet fluorescence microscopy, epifluorescence microscopy, multiphoton microscopy and confocal fluorescence microscopy.

[0273] 201. The analytical method according to any one of the preceding embodiments, wherein the fluorescence microscopy is fluorescence microscopy, preferably light sheet fluorescence microscopy.

[0274] 202. An analysis method according to any one of the preceding embodiments, wherein the method further comprises, after step i), step iii) dissecting a tissue region of interest; step iv) rehydrating the dissected tissue region of interest; and step v) further analyzing the dissected tissue region of interest.

[0275] 203. An analytical method according to embodiment 202, wherein in step v), the dissected tissue region of interest is further analyzed by antibody-based immunostaining, or by genetic analysis, preferably genetic analysis performed by RNAseq, or by proteomics, preferably proteomics performed by mass spectrometry.

[0276] 204. An analysis method according to any one of embodiments 202 or 203, wherein the dissected tissue region of interest includes metastases, preferably metastases with a size smaller than 200 tumor cells, more preferably metastases with a size smaller than 100 tumor cells, more preferably metastases with a size smaller than 75 tumor cells, more preferably metastases with a size smaller than 50 tumor cells, more preferably metastases with a size smaller than 25 tumor cells.

[0277] 205. A method of detecting metastasis, wherein the method comprises the method of analyzing animal tissue according to any one of the preceding embodiments.

[0278] 206. The method of detecting metastasis according to embodiment 205, which is a method of detecting metastasis in the animal tissue with single cell resolution throughout the animal tissue.

[0279] 207. The method for detecting metastasis according to any one of the preceding embodiments, wherein the animal tissue contains cancer metastasis, and wherein the target molecule labeled by the marker is a structure present in the cancer, preferably a protein.

[0280] 208. A method for analyzing the biodistribution of a biopharmaceutical drug, wherein the method comprises the method for analyzing animal tissue according to any of the preceding embodiments, wherein the animal has been treated with the biopharmaceutical drug defined in embodiment 161, wherein the animal tissue contains the biopharmaceutical drug, and wherein the biopharmaceutical drug is the target molecule labeled with the labeling agent.

[0281] 09. A method according to embodiment 208, wherein the biopharmaceutical drug is a therapeutic protein, preferably a therapeutic antibody.

[0282] 210. The method of embodiment 208, wherein the biopharmaceutical drug is a nanoparticle.

[0283] 211. A method for analyzing the biodistribution of nanoparticles, wherein the method comprises the method for analyzing animal tissue according to any one of the preceding embodiments, wherein the animal has been treated with nanoparticles, wherein the animal tissue contains the nanoparticles, and wherein the nanoparticles are the target molecules labeled with the labeling agent and / or are selected from nanoparticles conjugated with fluorescent dyes or nanoparticles that themselves have fluorescence.

[0284] 212. A method for analyzing the biodistribution of nanoparticles according to embodiment 211, wherein the nanoparticles carry drugs or are drugs.

[0285] 213. A method for studying neurodegeneration, wherein the method comprises the method of analyzing animal tissue according to any one of the preceding embodiments, wherein the animal tissue contains neurons.

[0286] 214. A method for studying neurodegeneration according to embodiment 213, wherein the neurons in the animal tissue have been fluorescently labeled, preferably by expressing a fluorescent protein.

[0287] 215. A method for studying neurodegeneration according to any one of the preceding embodiments, wherein the study of neurodegeneration comprises analysis of blebbing of neuronal axons.

[0288] 216. A method for studying neuroinflammation, wherein the method comprises the method of analyzing animal tissue according to any of the preceding embodiments, wherein the animal tissue contains neurons.

[0289] 217. A method for studying neuroinflammation according to embodiment 216, wherein the immune cells in the animal tissue have been fluorescently labeled, preferably by expressing a fluorescent protein.

[0290] 218. A method for studying neuroinflammation according to any of the preceding embodiments, comprising studying the activation of immune cells by analyzing the signal intensity and / or cell number of fluorescently labeled immune cells.

[0291] 219. A method for studying meningeal lymphatic vessels, wherein the method comprises the method of analyzing animal tissue according to any one of the preceding embodiments, and wherein the animal tissue preferably comprises an intact mouse head.

[0292] 220. A method for studying meningeal lymphatic vessels according to embodiment 219, wherein the meningeal lymphatic vessels are fluorescently labeled, preferably by a marker protein or by a tracer such as ovalbumin.

[0293] 221. A method for studying meningeal lymphatic vessels according to any one of the preceding embodiments, wherein the meningeal lymphatic vessels contain the target molecule.

[0294] 222. Use of cyclodextrin derivatives for improving the labeling of target molecules in fixed animal tissues or whole animal bodies with fluorescent dye-containing labeling agents.

[0295] 223. The use according to embodiment 222, wherein the fluorescent dye-containing labeling agent is a fluorescent antibody.

[0296] 224. The use according to embodiment 222 or 223, wherein the molecular weight of the fluorescent dye-containing marking agent is greater than 100 kDa.

[0297] 225. The use according to any one of embodiments 222 to 224, wherein the fluorescent dye-containing marking agent is as defined in any one of embodiments 80 to 115.

[0298] 226. The use according to any one of embodiments 222 to 225, wherein the cyclodextrin derivative is as defined in any one of embodiments 2 to 48.

[0299] 227. The use according to any one of embodiments 222 to 226, wherein the cyclodextrin derivative is not methyl-β-cyclodextrin having a degree of substitution of 1.8.

[0300] 228. The use according to any one of embodiments 222 to 227, wherein the cyclodextrin derivative is not methyl-β-cyclodextrin with a degree of substitution <2.0.

[0301] 229. The use according to any one of embodiments 226 to 228, provided that when R 2 , R 3 and R 6 When each is selected from H and CH3, then DS≥2.

[0302] 230. The use according to any one of embodiments 226 to 229, wherein

[0303] R 2 and R 6 is CH3, and R 3 is H; or

[0304] R 2 , R 3 and R 6 Each is independently selected from H and CH2CH(OH)CH3, and / or DS ≥ 0.9.

[0305] 231. The use according to any one of embodiments 222 to 229, wherein the cyclodextrin derivative is selected from (2-hydroxypropyl)-β-cyclodextrin, triacetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, heptath(2,6-di-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, γ-cyclodextrin and α-cyclodextrin; more preferably (2-hydroxypropyl)-β-cyclodextrin or heptath(2,6-di-O-methyl)-β-cyclodextrin; most preferably heptath(2,6-di-O-methyl)-β-cyclodextrin.

[0306] 232. The use of any one of embodiments 222 to 231, wherein the use is not a method of treatment of the human or animal body by surgery or therapy, nor a method of diagnosis performed on the human or animal body.

[0307] 233. A composition comprising an antibody and a cyclodextrin derivative, said antibody preferably having a molecular weight greater than 100 kDa.

[0308] 234. A composition according to embodiment 233, wherein the cyclodextrin derivative is as defined in any one of embodiments 2 to 48.

[0309] 235. The method according to embodiment 234, provided that when R 2 , R 3 and R 6 When each is selected from H and CH3, then DS≥2.

[0310] 236. The composition of embodiment 233 or 234, wherein:

[0311] R 2 and R 6 is CH3, and R 3 is H; or

[0312] R 2 , R 3 and R 6Each is independently selected from H and CH2CH(OH)CH3, and / or DS≥0.9.

[0313] 237. A composition according to any one of embodiments 233 to 236, wherein the antibody is a labeling agent containing a fluorescent dye, preferably as defined in any one of embodiments 80 to 115.

[0314] 238. A composition according to any one of embodiments 233 to 236, wherein the antibody is a first antibody, preferably as defined in any one of embodiments 172 to 178.

[0315] 239. The composition of any one of embodiments 233 to 238, further comprising one or more, preferably all, of the following components in a buffered aqueous solution, preferably phosphate buffered saline (PBS):

[0316] - animal serum, preferably mammalian serum, more preferably goat serum;

[0317] - a zwitterionic surfactant, preferably CHAPS or CHAPSO, more preferably CHAPS;

[0318] - a nonionic surfactant, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100;

[0319] - an organic solvent, preferably a water-miscible solvent, more preferably DMSO;

[0320] - an amino acid, preferably glycine.

[0321] 240. The composition of embodiment 239, wherein the components, if present in a buffered aqueous solution, have the following concentrations:

[0322] - Cyclodextrin derivatives: 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v;

[0323] - Animal serum: 0.5 to 12, preferably 0.75 to 10, more preferably 1 to 3% v / v;

[0324] - zwitterionic surfactant: 5 to 15, preferably 7.5 to 12.5, more preferably 10% w / v;

[0325] - nonionic surfactant: 0.5 to 4, preferably 1 to 3, more preferably 2% w / v;

[0326] - organic solvent: 5 to 20, preferably 7.5 to 15, more preferably 10% w / v;

[0327] - Amino acids: 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v;

[0328] And wherein the buffer concentration of the aqueous buffer is 0.05 to 0.2M, preferably 0.08 to 1.2M, more preferably 0.1M.

[0329] 241. The composition of any one of embodiments 233 to 240, comprising:

[0330] In 0.05 to 0.2 M, preferably 0.08 to 1.2 M, more preferably 0.1 M phosphate buffered saline

[0331] - 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v cyclodextrin derivative,

[0332] - 0.5 to 12, preferably 0.75 to 10, more preferably 1 to 3% v / v goat serum;

[0333] -5 to 15, preferably 7.5 to 12.5, more preferably 10% w / v CHAPS;

[0334] - 0.5 to 4, preferably 1 to 3, more preferably 2% w / v Triton X-100;

[0335] -5 to 20, preferably 7.5 to 15, more preferably 10% w / v DMSO; and

[0336] - 0.5 to 2, preferably 0.75 to 1.5, more preferably 1 % w / v glycine.

[0337] 242. The composition according to any one of embodiments 233 to 241, having a pH value of 7 to 7.4, preferably 7.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0338] Figure 1Development of wildDISCO and single antibody whole-mouse staining. (a) Structures of cyclodextrins (CDs) with different substituent groups, CD1 (methyl-β-cyclodextrin), CD2 (2-hydroxypropyl-β-cyclodextrin), CD3 (triacetyl-β-cyclodextrin), CD4 ((2-hydroxyethyl)-β-cyclodextrin), CD5 (heptakis(2,6-di-O-methyl)-β-cyclodextrin), CD6 (succinyl-β-cyclodextrin). (b) Supernatant cholesterol concentrations were measured on day 7 in 25 mg mouse liver sections with control, CD1, CD2, CD4, CD5, CD6, γ-cyclodextrin (CD7), and α-cyclodextrin (CD8) after incubation with different CD-containing buffers. (c) Methylene blue staining of a single mouse cerebral hemisphere after permeabilization with solutions containing different CDs. CD5 greatly enhances tissue permeabilization compared to the other substances, thereby facilitating dye migration. (d) Dynamic light scattering (DLS) detection of the size distribution of TH antibodies in solutions with and without CD5. (e) Dark coding shows the projections of pan-neuronally labeled PGP-9.5+ neurons at different z levels in a 2.0 cm thick whole mouse. (f, g) Details of the innervation of hard tissues (f, vertebrae) and soft tissues (g, adipose tissue). (h) Manually segmented vagus nerve (green) innervating the liver (cyan), spleen (magenta), intestine (red), and kidney (yellow), highlighted with specific pseudocolors. (i) Whole mouse stained with the lymphatic vessel marker LYVE1 (yellow). (j) Lymphatic element (LYVE1) staining was detected in the brain parenchyma of (i) mouse. (k) Mouse brain stained with two different lymphatic vessel markers (LYVE1 and podoplanin) allows identification of lymphatic endothelial cells in different brain regions.

[0339] Figure 2: Using wildDISCO to study the spatial relationships of different physiological systems. (a) Maximum intensity projection of a mouse stained with antibodies against the sympathetic nerve marker tyrosine hydroxylase (TH) (green) and the immune cell marker CD45 (magenta), showing the landscape of neural-immune interactions in visceral organs. (b) Branches of the sympathetic nervous system (TH, green) connect different regions of the intestine. CD45+ cells (magenta) are concentrated along various parts of the vagus nerve, especially in the inferior mesenteric plexus. (c) High-magnification view of the labeled area in (a), showing the colocalization of sympathetic nerve fibers and immune cells in the intestinal wall. (d) Maximum intensity projection of a whole mouse stained with TH (green) and LYVE1 (yellow). (eg) Representative 2D optical sections of hindlimb LNs stained with TH, PGP 9.5, CD45, Prox1, and LYVE1, as shown, showing that the LNs are innervated by peripheral nerves with immunomodulatory potential. (h, i) Representative 3D images of the myenteric neural grid network of WT and germ-free mice by immunostaining with an antibody against PGP9.5. (jk) Higher magnification views of the areas marked by blue and red boxes in (h) and (j), respectively. In germ-free mice, the myenteric neural grid network appears disorganized with fewer ganglia. (l) The density of PGP 9.5 myenteric plexuses was quantified. n = 5; mean ± SD **p < 0.01 (Student's t-test).

[0340] Figure 3 : Overview of wildDISCO immunostaining buffer and quantification of permeabilization effects.

[0341] (a) Illustration of the main chemical components involved in wildDISCO immunostaining buffer. (b) Figure 1 Cross-sections of the mouse brain along each dimension in c. (c) Methylene blue staining of a single mouse brain hemisphere and imaging of different brain cryosections after permeabilization with solutions containing different CDs. The results show that CD5 significantly enhances tissue permeabilization compared to other substances. n=4. (d) Figure 3 c Outline image along each cryosection of the mouse brain.

[0342] Figure 4 : WildDISCO immunostaining of PGP9.5 in whole mice. (a) Maximum projection of the peripheral nervous system of a 4-week-old mouse stained with PGP 9.5 antibody using light sheet microscopy. (b) Examples of PGP 9.5 positive staining in various organs (heart, spleen, liver, and intestine) with higher magnification areas. (f) Visualization of peripheral nerve innervation on multiple organs (adrenal glands (green), kidneys (magenta), and ureters (cyan)).

[0343] Figure 5 : WildDISCO immunostaining of LYVE1 in whole mice. Examples of optical sections of whole mice stained with LYVE1 antibody in different organs, liver (a), hind limb (b), adipose tissue (c), kidney (d), trachea (e), stomach (f), intestine (g), with higher magnification areas. (hi) 3D reconstructed view of the intestinal lymphatic network using Syglass reconstruction software.

[0344] Figure 6 : Nerve-immune cell interactions in the intestine. (a) 3D reconstruction of the intestinal Peyer's patches stained with CD45 (green) and innervated by TH+ sympathetic nerves (magenta), visualized using Syglass software. (b) Sympathetic nerve marker TH (green) and immune cell marker CD45 (magenta) on the intestinal wall, visualized using Imaris software.

[0345] Figure 7 : Nerve-lymphatic cell interactions in the intestine. (a) PGP9.5 nerve fibers (magenta) interact with Prox1 lymphatic vessels (green). (b) TH sympathetic nerves (green) and LYVE1 lymphatic vessels (magenta).

[0346] Figure 8 : Nerve-lymphatic cell interactions in the kidney. (a) TH sympathetic neurons (green) in the kidney innervate LYVE1 lymphatic vessels (magenta). (b) PGP9.5 pan-neuronal marker (green) binds to Prox1 lymphatic vessel marker (magenta).

[0347] Fig. 9 : Effects of the microbiota on mouse sympathetic nerves. (ab) Neural meshwork of intestinal muscles of WT and germ-free mice by immunostaining with antibodies against TH. Higher magnification views of the areas marked by white and yellow boxes, respectively.

[0348] Fig.10 : WildDISCO applied to a cancer metastasis model. Tyrosine hydroxylase TH is co-stained with breast cancer cells MDA-MB-231. Cancer cells are clearly visible around TH+ nerves.

[0349] Fig.11: WildDISCO shows the entire mouse arterial network. Alpha smooth muscle actin (α-SMA) was used to study the distribution of arteries throughout the body. Arteries with larger diameters branch into arterioles that run from the meninges through the corpus callosum (A). The distribution of arterioles in the eyes, olfactory bulbs, meninges, and brain is symmetrical and regular (B). Since α-SMA is a definitive marker for mature cardiac fibroblasts, called myofibroblasts, the coronary arteries and their associated branches are clearly visualized in the heart (C). α-SMA was detected in the liver sinusoidal spaces as well as in the portal and central veins (D). The splenic artery branches into arterioles, forming a reticular capillary network. (E) Cone-shaped vascular structures were also observed in the kidney and lung (F and G). Dorsal cross-sections of α-SMA-labeled mice show the distribution of arteries, especially arterioles in the spinal cord (H), in several organs.

[0350] Fig.12 : Schematic diagram of the wildDISCO pipeline (example order of steps (① to ⑥); preferred conditions for each step).

[0351] Fig.13 : Comparison of different clearing methods for whole-body antibody staining in mice. (a) Optical 2D light sheet microscopy images of the whole mouse body stained with synapsin 1 antibody by wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods, respectively. Scale bar, 5000 μm. (b) Representative 2D optical images of mouse hindlimb and spinal cord obtained by wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods. Scale bar, 300 μm. n=3. (c) Quantification of the depth of antibody penetration into mouse hindlimb and spinal cord by wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods.

[0352] Fig.14 : Comparison of different clearing methods for staining with synapsin-1. (ac) Representative 2D optical images of forelimb, liver, and kidney stained with synapsin-1 antibody by wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods, respectively. n = 3. (df) Quantification of the depth of synapsin-1 antibody penetration into mouse forelimb (d), liver (e), and kidney (f) using wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS, respectively.

[0353] Fig.15: Visualization and analysis of tumor-associated TLS in a tumor metastasis model using wildDISCO and deep learning. (a) 3D rendering of a mouse with 4T1 cell metastasis imaged using light sheet microscopy in a lateral abdominal view. TLS are detected and masked in magenta, tumor cells are masked in yellow, and the background color is cyan. Higher magnification views show details of the TLS. Scale bar, 2000 μm. (b–l) Example images of TLS in tumor-bearing mice stained with red CD23 (b, c) and green CD3 and magenta CD23 (d–g). TLS in primary tumors (h), intestines (i, j), and lungs (k, l) are masked in magenta. Scale bars: 500 μm (d, h, k); 150 μm (e, f, g, j); 400 μm (i) and 200 μm (l). (m–s) Quantification of the spatial correlation between TLS and metastasis in the whole mouse. (m) Quantification of whole-body metastasis volume in mice. (n) Quantification of metastasis density in the lung and intestine. n = 4 mice. Mean ± SD. (N) Quantification of TLS distribution throughout the mouse. n = 4 mice. Mean ± SD. (p) Quantification of TLS volume in mice. (q, r) Quantification of the distance to the nearest neighbor TLS (q), and the distance between a metastasis and the nearest TLS (r). (s) Metastasis volume to the nearest TLS. DETAILED DESCRIPTION

[0354] Definition and general techniques

[0355] Unless otherwise defined below, the terms used in the present invention should be understood according to their ordinary meanings known to those skilled in the art.

[0356] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. These publications, patents and patent applications mentioned herein are preferably identified by their first author name and year of publication, or by numbers. For each reference identified in this manner, the corresponding reference, including the specific source of the publication (e.g., name and volume of a scientific journal, etc.), can be found in the "References" section.

[0357] Unless otherwise stated, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0358] The term "fluorescent dye" as used herein is not particularly limited. For example, the fluorescent dye can be a fluorescent protein or a synthetic compound, such as a synthetic organic compound. Preferably, the fluorescent dye used according to the present invention is capable of emitting fluorescence in the red or infrared range, more preferably in the far-red or near-infrared range. The preferred wavelength of the emission maximum of the fluorescent dye used according to the present invention is as shown in the preferred embodiment of the present invention. Non-limiting examples of fluorescent dyes that can emit fluorescence in the far-infrared or near-infrared range and can be used according to the present invention are known in the art and have been reviewed, for example, in Hong et al. (2017), Near-infrared fluorophores for biomedical imaging. Nature Biomedical Engineering 1, 0010, which is incorporated herein by reference in its entirety for all purposes. Fluorescent dyes that are capable of emitting fluorescence in the far-red or near-infrared range and that can be used according to the present invention are commercially available and preferably include, for example, ATTO dyes, such as ATTO Rho 13, ATTO 594, ATTO 550, ATTO 610, ATTO 620, ATTO Rho 14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa 12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, and ATTO 740, and Alexa Fluor® dyes, such as Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, and Alexa Fluor® 760. 790, and Cy dyes such as Cy7, Cy5 and Cy3. Dyes with emission maxima at 488 nm, 555 nm and 568 nm are also known in the art and can also be used in the methods of the present invention.

[0359] It is also contemplated that labeling is performed using fluorescent dye-containing labeling agents having a molecular weight of 100 kDa or less. That is, while the present invention is particularly advantageous for improving or enhancing the labeling of target molecules in fixed animal tissues or whole animals using fluorescent labeling agents (e.g., antibodies) having a molecular weight greater than 100 kDa, it is also contemplated that comparable improvements can be achieved by using the cyclodextrin derivatives described herein to improve labeling using agents having a molecular weight of 100 kDa or less (e.g., antibody fragments conjugated to fluorescent dyes or other dyes).

[0360] The animal tissues that can be used in the purposes and methods of the present invention are not particularly limited. They can be from any animal species. In a preferred embodiment of the present invention, the animal tissue can be tissue from a non-human mammal or from a human. Preferably, the animal tissue from a non-human mammal is from a rodent, more preferably from a mouse. More preferably, the animal tissue is a whole mouse. In a preferred embodiment according to the present invention, the animal tissue can be a whole organ or part thereof, preferably a human organ or part thereof. The animal tissue can contain recombinantly expressed fluorescent proteins (such as GFP, YFP and mCherry), which can be used as target molecules. For example, the animal from which the animal tissue is obtained can be an animal (such as a mouse) that has been transplanted with cancer cells expressing such recombinant fluorescent proteins. The fixed animal tissue used in the purposes and methods of the present invention preferably has a size of at least 1 cm long × at least 1 cm wide × at least 1 cm high.

[0361] The decolorization step of the preparation method of the present invention uses fixed animal tissue. In a preferred embodiment according to the present invention, the preparation method of the present invention starts from the decolorization step and does not include the fixation of animal tissue. Therefore, in a preferred embodiment, all methods and uses of the present invention may preferably be methods for treating the human or animal body not by surgery or therapy, nor are they diagnostic methods implemented on the human or animal body. In a relevant preferred embodiment according to all other embodiments of the present invention, the method or use of the present invention is an in vitro method and use. Therefore, the method of the present invention may preferably be carried out in vitro of a living animal.

[0362] Those skilled in the art can easily identify animal tissues suitable for fixation in the method of the present invention. For example, for PFA fixation, mice can be deeply anesthetized using a combination of midazolam, medetomidine and fentanyl (MMF) (e.g., 1 mL / 100 g body weight for mice; intraperitoneal injection), and then perfused intracardially with heparinized 0.1 M PBS at room temperature (10 U / mL heparin, Ratiopharm; using Leica Perfusion One system, pressure of 100-125 mmHg) for 5-10 minutes until the blood is washed out. This process is followed by fixation with, for example, 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) (Morphisto, 11762.01000) for 10-20 minutes. If staining of blood vessels is desired, tissues from animals such as mice (e.g., whole mice) can be perfused intracardially with 0.5 mg FITC-conjugated lectin (EY Laboratories, F-2101-5) in 20 ml PBS (without heparin) prior to PFA fixation. Alternatively, for PaXgene fixation, mice can be deeply anesthetized with a combination of midazolam, medetomidine, and fentanyl (MMF) (e.g., 1 mL / 100 g body weight for mice; intraperitoneal) and then perfused intracardially with heparinized 0.1 M PBS (10 U / mL heparin, Ratiopharm; using a Leica Perfusion One system at 100-125 mmHg) for 5-10 minutes at room temperature until blood is washed out. This procedure is followed by fixation by injection of 40-50 ml PaXgene fixative. If PaXgene-fixed animals need to be stored prior to further processing, tissues are stored in PaXgene stabilization solution. If staining of blood vessels is desired, animal tissue such as mice (e.g., whole mice) can be intracardially perfused with 20 ml PBS (without heparin) containing 0.5 mg FITC-conjugated lectin (EY Laboratories, F-2101-5) prior to PFA fixation. Subsequently, the skin can be carefully removed, or left intact if the animal is nude (no fur), and the body can be post-fixed in 4% PFA at 4°C for 1 day and then transferred to 0.1 M PBS. The method of the invention can be started immediately, or the whole mouse body can be stored, preferably in PBS at 4°C for up to 4 weeks, or in PBS containing 0.05% sodium azide (Sigma, 71290) for up to 6 months.

[0363] As used herein, "animal tissue for fluorescence microscopy" and the like terms are intended to indicate that the corresponding animal tissue is suitable for fluorescence microscopy.

[0364] Similarly, the term "removing heme" in relation to a solution refers to any solution suitable for removing heme. According to the present invention, the removal of heme is not limited to a particular mechanism, as long as it can remove heme from tissue and / or decolorize heme. For example, amino alcohols suitable for removing heme can be used, such as N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, for example as shown in the preferred embodiment. This amino alcohol competes with hemoglobin for heme binding and can therefore be used to remove heme from hemoglobin and tissue in the tissue. Alternatively, benzoyl peroxide can be used.

[0365] The term "labeling agent containing a fluorescent dye" used in the present invention is not particularly limited, as long as it is suitable for labeling a target molecule in a decolorized fixed animal tissue, can bind to the target molecule, and has a molecular weight greater than 100 kDa (e.g., greater than or equal to 110 kDa, greater than or equal to 120 kDa, greater than or equal to 130 kDa, or greater than or equal to 140 kDa). In a preferred embodiment, the labeling agent containing a fluorescent dye is an antibody conjugated with the fluorescent dye.

[0366] As used herein, the term "target molecule" refers to any target molecule in a tissue. It should be understood that for specific applications of the method of the present invention, such as biomedical applications, appropriate target molecules can be selected. These target molecules can be, for example, endogenous molecules (e.g., marker proteins for diseases such as cancer) or recombinant molecules, such as recombinant proteins, of animals. For example, in a preferred embodiment according to the present invention, if the animal from which the animal tissue is obtained is an animal (e.g., a mouse) transplanted with cancer cells expressing such recombinant fluorescent proteins, such fluorescent proteins can be target molecules. Alternatively, the target molecule can be any other structure (e.g., an exogenous molecule) present in fixed animal tissues, such as proteins, lipids, DNA or RNA present in the fixed animal tissues, more preferably proteins present in the fixed animal tissues, such as therapeutic antibodies or first antibodies that bind to tissue antigens in fixed animal tissues.

[0367] The first antibody that can be used in the present invention is not particularly limited. In one embodiment, the first antibody is selected from the group consisting of anti-tyrosine hydroxylase antibody, anti-PGP 9.5 antibody, anti-S100β antibody, anti-neurofilament M antibody, anti-α smooth muscle actin antibody, anti-type IV collagen antibody, anti-Prox1 antibody, anti-LYVE1 antibody, anti-Iba1 antibody and anti-CD45 antibody. Preferably, the first antibody is not a rabbit anti-neurofilament NF-M antibody.

[0368] It should also be understood that the term "labeled target molecule" as used herein includes the possibility that the method of the present invention can label more than one target molecule. Therefore, in a preferred embodiment of the present invention, more than one target molecule, such as two or three target molecules, are labeled. For example, in a preferred embodiment according to the present invention, if the animal from which the animal tissue is obtained is an animal (e.g., a mouse) transplanted with cancer cells expressing such a recombinant fluorescent protein, such a fluorescent protein can be the first target molecule, and the anti-cancer biopharmaceutical drug (e.g., an anti-cancer therapeutic antibody) administered to the animal can be the second target molecule. Therefore, in a preferred embodiment such as the present embodiment, the metastasis detection method according to the present invention and the method for analyzing the biodistribution of the biopharmaceutical drug can be performed together.

[0369] The term "perfusion under pressure" as used herein refers to a measurable pressure at the tissue entrance. Pressure can be measured by any method known in the art. Preferably, the pressure is measured with a manometer, more preferably with a Kkmoon digital manometer pressure gauge manometer (HT-1891). When using a Kkmoon digital manometer pressure gauge manometer (HT-1891), a 2-head connector (B.Braun Discofix® C Dreiwegehahn, 16494C) can be inserted into the pumping channel and connected to the pressure gauge. The pumping channel can be set with a transcardial perfusion needle (Leica, 39471024), and the pressure (when the reading is stable) can be measured at the pumping speed used by the method.

[0370] The "permeabilization solution" referred to herein refers to a solution suitable for permeabilization of animal tissues. Such solutions are known in the art and can be easily selected by a person skilled in the art and include, for example, suitable surfactants for permeabilization, such as CHAPS (3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonate; CAS No.: 75621-03-3), CHAPSO (3-([3-cholamidopropyl]dimethylammonium)-2-hydroxy-1-propanesulfonate; CAS No.: 82473-24-3), IGEPALCA-630 (octylphenoxy poly(ethyleneoxy)ethanol; CAS No.: 68412-54-4; linear molecular formula: (C2H4O) n C 14 H 22 O (e.g., average n = 9)) and / or Triton X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol; CAS No.: 9002-93-1; linear formula: tert-octyl-C6H4-(OCH2CH2) x OH (e.g., average x = 9.5)).

[0371] The "labeling solution", "immunostaining solution" or "immunostaining buffer" referred to herein refers to a solution containing a labeling agent containing a fluorescent dye, unless otherwise specified. Typically, the labeling solution contains one or more surfactants, an organic solvent and a labeling agent.

[0372] Advantageously, the labeling solution comprises one or more, preferably all, of the following components in a buffered aqueous solution, preferably phosphate buffered saline (PBS): animal serum, preferably mammalian serum, more preferably goat serum; a zwitterionic surfactant, preferably CHAPS or CHAPSO, more preferably CHAPS; a nonionic surfactant, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100; an organic solvent, preferably a water-soluble solvent, more preferably DMSO; and / or an amino acid, preferably glycine.

[0373] Preferably, the permeabilization solution and / or labeling solution used according to the invention further comprises an agent suitable for extracting cholesterol from biological membranes. Without wishing to be bound by any theory, it is hypothesized that poor extraction of cholesterol from cell membranes may be a limiting factor for permeabilization in prior art solutions. 18 Suitable agents for extracting cholesterol from biological membranes include, for example, cyclodextrin derivatives. Preferred cyclodextrin derivatives are summarized in Table 1 below.

[0374] Table 1 Cyclodextrin derivatives

[0375]

[0376]

[0377] As used herein, the term "total degree of substitution" or "TDS" refers to the number of non-hydrogen substituents R per mole of substituted cyclodextrin. 2 , R 3 , R 6 TDS can be measured by methods known in the art, such as 1 H NMR, ESI-MS or MALDI-TOF-MS. For a substituted cyclodextrin whose non-hydrogen substituent is represented by R, TDS can be calculated based on the number average molecular weight Mn according to the following formula:

[0378] TDS = [M n –M(unsubstituted cyclodextrin)] / [M(R)-1].

[0379] The term "degree of substitution" or "DS" as used herein refers to the number of non-hydrogen substituents R per mole of glucopyranose units in a cyclodextrin. 2 , R 3 , R 6The DS can be determined by methods known in the art. For a certain substituted cyclodextrin, the DS can be calculated according to the total degree of substitution (TDS) defined above according to the following formula:

[0380] DS = TDS / m

[0381] where m is the number of glucopyranose units in the cyclodextrin molecule (for α-cyclodextrin, m = 6; for β-cyclodextrin, m = 7; for γ-cyclodextrin, m = 8).

[0382] A preferred cyclodextrin derivative is a cyclodextrin derivative with m=7, i.e., β-cyclodextrin. The cyclodextrin derivative is preferably selected from (2-hydroxypropyl)-β-cyclodextrin, triacetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, heptath(2,6-di-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, γ-cyclodextrin and α-cyclodextrin; more preferably (2-hydroxypropyl)-β-cyclodextrin or heptath(2,6-di-O-methyl)-β-cyclodextrin; most preferably heptath(2,6-di-O-methyl)-β-cyclodextrin. The cyclodextrin derivative is preferably not methyl-β-cyclodextrin, for example, not methyl-β-cyclodextrin with a degree of substitution <2.0, for example, 1.8. A particularly preferred cyclodextrin derivative is one in which R 2 , R 6 =CH3 and R 3 =H, more preferably heptakis(2,6-di-O-methyl)-β-cyclodextrin (ie, CD5); or wherein R 2 , R 3 and R 6 Selected from H and CH2CH(OH)CH3, more preferably (2-hydroxypropyl)-β-cyclodextrin (such as CD2); most preferably heptakis(2,6-di-O-methyl)-β-cyclodextrin.

[0383] Preferably, the cyclodextrin derivative used in the present invention is capable of extracting cholesterol from mouse liver tissue, so that the cholesterol concentration after 7 days of incubation is ≥14, preferably ≥30, more preferably ≥50, and most preferably ≥70 μM, measured under the conditions described in the following example "Screening of cyclodextrin-containing buffers for cholesterol extraction".

[0384] Preferably, the permeabilization solution used according to the invention also contains an agent that relaxes the collagen network, such as trans-1-acetyl-4-hydroxy-L-proline. It should be understood that if a permeabilization solution is used in a particular step of the method of the invention, this does not exclude that other solutions (such as solutions used in previous steps of the method) may also contribute to permeabilization and improve permeabilization. For example, a solution used to remove heme can be a solution that contributes to permeabilization.

[0385] The solutions or compositions described herein, particularly the labeling solutions, typically contain a zwitterionic surfactant, such as CHAPS or CHAPSO, or a combination of a zwitterionic surfactant and a nonionic surfactant, such as Triton X-100 or IGEPAL CA-630. Each of these surfactant types is believed to enhance permeabilization; and their combination is believed to provide a synergistic enhancement, for example, for the following reasons:

[0386] Surfactant properties: Zwitterionic surfactants (such as CHAPS) are both positively and negatively charged, so they are gentler on proteins and less likely to denature. Nonionic surfactants (such as Triton X-100) are equally gentle, but have different affinities for various proteins and lipids. Therefore, this combination can solubilize a wider range of biomolecules.

[0387] Micelle formation: Surfactants work by forming micelles around the hydrophobic (water-insoluble) portion of the molecule, making it soluble in aqueous solutions. The size and properties of the micelles formed by different surfactants can vary, allowing them to more effectively solubilize different types of molecules when used together.

[0388] Protein solubilization: Different proteins have different conformations and hydrophobic regions. Using a combination of these two surfactants is thought to be more effective in solubilizing various proteins without causing denaturation, as the surfactants can complement each other to stabilize different proteins.

[0389] According to the present invention, the measurement of tissue volume may be performed by any suitable method known in the art. Preferably, tissue volume is measured by measuring the volume displacement of fluid in the tissue, for example in a suitable cylinder.

[0390] Preferably, the method of the present invention further comprises a blocking step. The blocking step is to treat the fixed animal tissue with a blocking solution before the labeling step. The "blocking solution" referred to herein is suitable for blocking non-specific antigen binding of antibodies and may include, for example, animal serum. The animal serum may be mammalian serum, preferably goat serum or donkey serum, more preferably goat serum; and may be present in a concentration of, for example, 1 to 15, preferably 3 to 10, more preferably 3% v / v. The blocking solution may also include a surfactant, such as a nonionic surfactant, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100. The nonionic surfactant may be present in a concentration of, for example, 0.5 to 4, preferably 1 to 3, more preferably 2% w / v. The components of the blocking solution are preferably present in a buffered aqueous solution, preferably phosphate buffered saline (PBS), which optionally has a buffer concentration of 0.05 to 0.2M, preferably 0.08 to 1.2M, more preferably 0.1M.

[0391] In a preferred embodiment, treatment with a permeabilizing solution is performed simultaneously with treatment with a blocking solution, for example by providing a permeabilizing solution that is also a blocking solution, preferably comprising animal serum and a surfactant as described above.

[0392] A particularly preferred method according to the invention comprises the following steps, preferably in the following order:

[0393] - destaining, permeabilization and blocking steps, which are performed by treating the fixed animal tissue with one or more solutions of heme removal, permeabilization and blocking;

[0394] - Labeling the target molecule in the fixed animal tissue with a labeling solution, wherein the labeling solution comprises a cyclodextrin derivative and a labeling agent containing a fluorescent dye capable of binding to the target molecule, wherein the labeling agent preferably has a molecular weight greater than 100 kDa, so as to obtain the fixed animal tissue labeled with the labeling agent containing the fluorescent dye.

[0395] An even more preferred method according to the invention comprises the following steps, preferably in the following order:

[0396] - destaining, permeabilization and blocking steps, which are performed by treating the fixed animal tissue with one or more solutions of heme removal, permeabilization and blocking;

[0397] - contacting the fixed animal tissue with a first labeling solution comprising a cyclodextrin derivative and a first antibody capable of binding to a structure present in the fixed animal tissue, preferably a protein, a lipid, a DNA or an RNA, more preferably a protein present in the fixed animal tissue;

[0398] - Labeling the first antibody that has bound to the structure in the fixed animal tissue with a second labeling solution comprising a cyclodextrin derivative and a labeling agent containing a fluorescent dye, wherein the labeling agent is capable of binding to the first antibody and preferably has a molecular weight greater than 100 kDa, to obtain a fixed animal tissue labeled with the labeling agent containing the fluorescent dye.

[0399] The heme-removing solution, permeabilization and blocking solution are preferably the same solution ("pretreatment solution"), and preferably have the composition as described above for the blocking solution.

[0400] The first labeling solution and the second labeling solution preferably have the composition of the labeling solution as described above. For example, the first labeling solution may have the composition of the labeling solution as described above, wherein the antibody is a first antibody (e.g., an antibody capable of binding to a structure present in fixed animal tissue); the second labeling solution may have the composition of the labeling solution as described above, wherein the antibody is a fluorescent dye-containing labeling agent capable of binding to the first antibody (e.g., an antibody conjugated to a fluorescent dye). The composition suitable for use as the labeling solution described herein preferably has a pH value of 7 to 7.4, more preferably 7.2.

[0401] "Clearing solution" as referred to herein refers to a solution suitable for clearing animal tissue. Such solutions are not particularly limited as long as they contain an organic solvent. It should be understood that those skilled in the art can easily select such organic solvents to make them compatible with the method of the present invention, for example based on their electromagnetic absorption / emission spectra (particularly their lack of fluorescence emission in the visible, red and near infrared ranges). Preferably, the clearing solution comprising an organic solvent has a refractive index similar to that of the tissue (e.g., bone) of the animal, as reflected in the preferred embodiment. Such clearing solutions are particularly advantageous for the clearing of tissues. Examples of preferred organic solvents that can be used for such clearing solutions of the present invention are, for example, solvents comprising benzyl alcohol, benzyl benzoate and diphenyl ether, solvents comprising ethyl cinnamate and solvents comprising allyl cinnamate.

[0402] The method for measuring the refractive index is well known in the art. The refractive index value referred to herein is the value measured at room temperature (ie 25° C.) and normal atmospheric pressure (ie 760 mmHg).

[0403] The "additional organic solvent" mentioned in the present invention is not particularly limited. It is understood that a person skilled in the art will select such solvents so that they are suitable for dehydration. Examples of such solvents are THF, dichloromethane and 1,4-dioxane. For example, according to the present invention, the perfusion of the additional organic solvent preferably increased with a gradient of 0 vol% to 100 vol% can be the perfusion of THF with a gradient of 0 vol% to 100 vol%, followed by incubation with dichloromethane. Alternatively, in all embodiments according to the present invention, dichloromethane can be replaced with 1,4-dioxane.

[0404] According to all further embodiments of the invention, further improvements may be achieved by adding a decalcification step. Such a step will further improve the transparency of the bone. Decalcification chemicals are known and include, for example, solutions containing EDTA, preferably also containing NaHCO3. Preferably, the pH value of the solution used for decalcification is 8-9. Such a decalcification step is performed before the decolorization step, or if no decolorization step is performed, it is performed before the labeling step.

[0405] The term "existing in the fixed animal tissue" used herein in relation to a structure refers to a structure existing in the fixed animal tissue. The term does not mean that the structure must exist within a cell, but also includes the possibility that the structure exists on or outside the cell of the fixed animal tissue, for example, on the cell surface of the fixed animal tissue or in the extracellular matrix of the fixed animal tissue.

[0406] The term "therapeutic antibody" as used herein refers to any therapeutic antibody and therapeutic antibody fragment known in the art. In addition, the term is not limited to therapeutic antibodies and therapeutic antibody fragments, but also includes conjugates, such as antibody drug conjugates.

[0407] The term "small molecule" as referred to herein has the meaning known in the art. Typically, small molecules used according to the present invention have a molecular weight < 900 Daltons.

[0408] According to the present invention, each occurrence of the term "comprising" may optionally be replaced by the term "consisting of".

[0409] The invention is illustrated by the following non-limiting examples.

[0410] Example

[0411] Unless otherwise stated, the following methods were used in the Examples.

[0412] Animals involved in the study

[0413] We used the following mixed-sex animals in the wildDISCO study: 4-week-old wild-type mice (C57BL / 6J, CD1, and Balb / c) purchased from Charles River Laboratories. Animals were housed on a 12 / 12 h light / dark cycle with ad libitum access to food and water. Temperature was maintained at 18–23 °C and humidity at 40–60%. Age- and sex-matched C57BL / 6J germ-free mice were purchased from the Technical University of Munich (Institute of Nutrition and Health, Core Facility Gnotobiology) and housed in sterile isolators. The absence of bacteria in germ-free mice was confirmed by microbiological culture, and mice were then used for further experiments. Each antibody was successfully replicated in at least five mice and in at least three different humans. Animal experiments were performed according to the institutional guidelines of the Ludwig Maximilian University of Munich and the Helmholtz Munich Center German Mouse Clinic after approval from the ethical review board of the Upper Bavarian State Government (Regierung von Oberbayern, Munich, Germany).

[0414] Screening of Cyclodextrin-Containing Buffers for Cholesterol Extraction

[0415] Cholesterol extraction was measured using the Cholesterol / Cholesteryl Ester-GloTM Assay (Promega, Madison, MD, USA). 25 mg of PFA-fixed mouse liver was incubated in 3 ml of 1% w / v antibody buffer containing different cyclodextrins: 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich, H107-100G, LOT WXBC6699V), methyl-β-cyclodextrin (Sigma-Aldrich, 332615-25G, LOT STBK8343), (2-hydroxyethyl)-β-cyclodextrin (Sigma-Aldrich, 389137-10G, LOT TMKBZ6644V), triacetyl-β-cyclodextrin (Sigma-Aldrich, 332623-10G, LOT STBJ9765), succinyl-β-cyclodextrin (Sigma-Aldrich, 85990-500MG, LOT BCCB7898) and heptakis (2,6-di-O-methyl)-β-cyclodextrin (Sigma-Aldrich, 39915-1G, LOT BCCF6041), γ-cyclodextrin (Sigma-Aldrich, C4892-5G, LOT TSLBJ8855V), α-cyclodextrin (Sigma-Aldrich, 779008-100G, LOT BCCJ0084). The assay was performed at different time points (2 days, 3 days, 5 days and 7 days). A 5 ul aliquot of the supernatant was diluted 10 times in cholesterol lysis solution and incubated at 37°C for 30 minutes. The cholesterol detection reagent was then added to the sample and incubated at room temperature for 60 minutes. The values ​​were measured using a Centro LB96 plate reading photometer (Berthold, Bad Wildbad, Germany).

[0416] Evaluation of the effects of buffers containing different cyclodextrins on antibody stability

[0417] The homogeneity of antibodies in buffers containing different cyclodextrins, in other words, antibody aggregation, was measured by dynamic light scattering (DLS). TH primary antibody was selected to evaluate antibody stability and homogeneity. TH antibody (Millipore, AB152) (Mw: 150 kDa, concentration: 10 g / l) was dissolved in buffer containing and without heptakis(2,6-di-O-methyl)-β-cyclodextrin (Sigma-Aldrich, 39915-1G) (1% w / v) at room temperature. After 7 days of incubation, the buffer solution was diluted and then measured in a folded capillary cell (DTS 1070) using a Zetasizer Nano ZS (Malvern, Worcestershire, UK). The samples were measured three times, with six sub-runs each time. The temperature was set to 25°C.

[0418] Using methylene blue to measure the permeabilization ability of buffers containing different cyclodextrins on hemibrain

[0419] Mouse hemibrains were incubated with 45 mL of various cyclodextrin buffers at 1% (w / v) at 37°C for 3 days. After washing twice with PBS, the samples were added with 45 mL of 0.03% methylene blue and incubated overnight at 37°C. To determine the efficacy of methylene blue staining after incubation with different CD buffers, the samples were cut in half along the midline to assess the efficacy of staining the inner tissue. Camera images of the samples were analyzed using ImageJ to draw contour maps and quantify pixels at threshold grayscale values.

[0420] Perfusion and whole body fixation of mice

[0421] Mice were deeply anesthetized with (0.05 mg / kg fentanyl, 0.5 mg / kg medetomidine, and 5 mg / kg midazolam intraperitoneal injection) and perfused intracardially with heparinized 0.01 M PBS (final heparin concentration 10-25 U / ml, Ratiopharm, N68542.03; perfusion volume 12 ml / min, using an ISMATEC peristaltic pump system). After flushing the blood of the mice for 5-10 minutes, they were perfused with 4% paraformaldehyde (PFA) in 0.01 M PBS (Morphisto, 11762.01000) for 10-20 minutes. After fixation in 4% PFA at 4°C for 6 hours, the mouse bodies were skinned and transferred to 0.01 M PBS.

[0422] wildDISCO whole-body immunostaining, PI labeling, and tissue clearing

[0423] The wildDISCO whole-body immunostaining protocol is based on a setup that pumps pretreatment solutions and immunostaining buffer into the mouse heart and vascular system to perfuse the entire body. The pumping setup has been described previously. 5,15. Briefly, after PFA fixation and two washes with 0.1 M PBS for 30 min, the mouse body was placed in a 300 ml glass chamber and a perfusion needle was inserted into the mouse heart through the same hole as for PFA perfusion. Then, the perfusion needle was connected to an ISMATEC peristaltic pump (REGLO Digital MS -4 / 8 ISM 834; reference tube, SC0266), which maintained the pressure at 160-230 mmHg (45-60 rpm) and was used to establish transcardiac circulation. The pump was equipped with two channels. One channel was used to pump the solution into the heart and circulate it in the mouse body, while the second channel collected and circulated the solution leaving the mouse body. In the first channel, a 1 ml syringe tip (Braun, 9166017V) was used to connect the perfusion needle (Leica, 39471024) and the reference tube (Ismatec Reglo, SC0266), which came from the pump and was set to circulate the solution from the heart to the vascular system. As the second channel allows solution recirculation, the inflow tube was immersed in the solution chamber of the glass chamber. After the pump and channels were set up, the needle was fixed with super glue (Pattex, PSK1C) to ensure continuous and stable perfusion. All the following perfusion steps were performed using the setup described above.

[0424] The mice were perfused with 0.1 M PBS overnight at room temperature, and then perfused with a decalcification solution containing 10 w / v% EDTA (Carl Roth, 1702922685) in 0.1 M PBS for 2 days, and all bones were decalcified at room temperature with sodium hydroxide (Sigma-Aldrich, 71687) adjusted to pH 8-9. The mice were then perfused with 0.1 M PBS three times, each wash for 3 hours. Next, the mice were perfused with a permeabilization and blocking solution containing 10% goat serum and 2% Triton X-100 in 0.1 M PBS for 1 day. The mice were then perfused with primary antibodies TH (Millipore, AB152), PGP9.5 (Proteintech, 14730-1-AP), LYVE1 (Thermo Fisher Scientific, 14-0443-82), CD45 (BD Biosciences, 550539), PROX1 (Abcam, ab101851), Podoplanin (Abcam, ab109059) (25 µg in 250 ml, diluted 1:10,000) or 290 µl PI (stock concentration 1 mg ml-1) and incubated for 7 days with 250 ml immunostaining buffer containing 3% goat serum, 10% CHAPS, 2% Triton X-100, 10% DMSO, 1% glycine, 1% heptadecan (2,6-di-O-methyl)-β-cyclodextrin in 0.1 M PBS. The mice were then washed three times with 0.1 M PBS for 12 hours each at room temperature. Then, the mouse bodies were perfused with Alexa fluorescent dye-conjugated secondary antibodies: AlexaFluor 647 goat anti-rabbit IgG antibody (Thermo Fisher Scientific, A-21245) or Alexa Fluor 647 goat anti-rat IgG antibody (Thermo Fisher Scientific, A-21247) (25 µg in 250 ml, diluted 1:10,000) in immunostaining buffer at room temperature for 7 days. The mouse bodies were washed three times with 0.1 M PBS for 12 hours each time.

[0425] After completing the immunostaining steps, transfer the mice to a fume hood and clear them using the 3DISCO passive whole-body clearing protocol reported previously. 9Briefly, the mouse body was placed in a 300 ml glass chamber and immersed in the following gradient of THF (tetrahydrofuran, Roth, CP82.1) in 200 ml distilled water under gentle shaking: (50% × 1, 70% × 1, 80% × 1, 100% × 2, 12 h for each step), then immersed in dichloromethane (DCM, Sigma, 270997) for 3 h, and finally immersed in BABB solution (benzyl alcohol + benzyl benzoate 1:2, Sigma, 24122 and W213802) until the body was optically transparent.

[0426] Light sheet microscopy imaging

[0427] Image stacks were acquired using a Blaze ultramicroscope (LaVision BioTec GmbH, version 7.3.2) with an axial resolution of 4 μm and the following filter sets: ex 470 / 40 nm, em 535 / 50 nm; ex 545 / 25 nm, em 605 / 70 nm; ex 640 / 40 nm, em 690 / 50 nm. The entire body of the mouse was scanned individually using an ultramicroscope Blaze light sheet microscope 4× objective (Olympus XLFLUOR 4× corrected / 0.28 NA [WD = 10 mm]). We used 9 × 23 tile scans to cover the entire mouse with a 20% overlap and separated imaging to a depth of 10 mm from the ventral and dorsal surfaces, covering the entire body volume with a Z step size of 10 µm. The light sheet width was reduced to 60% to achieve maximum illumination of the field of view, and the exposure time was set to 120 ms. The laser power was adjusted as a function of the fluorescence signal intensity to avoid saturation. The acquired raw image TIFF was processed using the Fiji stitching plug-in (http: / / www.discotechnologies.org / ).

[0428] Virtual Reality (VR) Headset Operation

[0429] A Virtual Reality (VR) headset is required to watch the videos. To visualize these videos, a VR video player needs to be installed on a VR device or PC. Videos played in VR need to have "_360" added to the end of the file name and be set to "360° / 3D" view in the VR player for an immersive experience.

[0430] Reconstruction and quantification of whole body scans

[0431] Detailed step-by-step instructions for image data stitching and volume fusion were provided previously 15Briefly, image stacks were recorded using ImSpector software (LaVision BioTec GmbH) and saved in TIFF format for each channel separately. Scanned mouse ventral and dorsal image data were first stitched using the Fiji stitching plugin, and then the volumes were fused using Vision4D (v3.5 × 64, Arivis AG, version 3.4.0). To improve the accuracy of volume fusion, alignment was performed by manually selecting 3 to 4 anatomical landmarks from the overlapping regions. Representative images were created using Imaris (Bitplane AG, version 9.6.0) and Vision4D for 3D volume reconstruction, maximum intensity projection, and deep color rendering. To isolate specific tissue regions, the Imaris surface tool was used manually with the option of masking channels with pseudocolor selected. After manual segmentation, the region was visualized in 2D slices using the OrthoSlicer tool. Virtual reality (VR) images and movies were generated using Syglass software (IstoVisio, Inc, version 1.7.2). To quantify myenteric plexus in the duodenum of germ-free and wild-type mice, five 200 μm × 200 μm × 200 μm cubic volumes along the portal triads were randomly selected from 3D images reconstructed in Imaris. The length of PGP 9.5-positive myenteric plexus in each cubic volume was traced using Imaris Filament Tracer.

[0432] Quantification

[0433] Data are presented as mean ± SD. Statistical analysis was performed using Prism GraphPad software v.8 with a 95% confidence interval. P values ​​were calculated using a two-tailed unpaired t-test to compare data between two groups. P values ​​< 0.05 were considered statistically significant.

[0434] Example 1 :Cholesterol extraction; methylene blue permeation into the whole mouse brain; preventing the formation of antibody aggregates

[0435] The present inventors investigated β-cyclodextrin variants ( Figure 1 a) Potential to promote cholesterol extraction from fixed samples and in combination with CHAPS and Triton X-100 surfactants to enhance permeabilization ( Figure 3 a). Using the cholesterol / cholesterol ester-glo assay to assess cholesterol extraction, the inventors found that heptakis(2,6-di-O-methyl)-β-cyclodextrin (CD5) extracted most of the cholesterol from mouse liver tissue after 7 days ( Figure 1b). CD2 (2-hydroxypropyl-β-cyclodextrin) also showed good cholesterol extraction ability.

[0436] Adding CD5 to the permeabilization reaction allowed methylene blue to quickly and evenly permeate throughout the mouse brain within 12 hours ( Figure 1 c and Figure 3 b).

[0437] It has been previously reported that cyclodextrins can stabilize proteins in solution by preventing aggregation 19 Therefore, dynamic light scattering (DLS) was used to measure the size of antibodies in antibody solutions. After 7 days at room temperature, the antibody showed two peaks in the DLS data for the solution without CD5, one at 11.5 nm, which may be the antibody monomer, and the other peak of larger size, which most likely corresponds to different aggregation states. Adding CD5 prevents the formation of aggregates ( Figure 1 d).

[0438] Example 2 :Improving the uniformity and depth of whole-body antibody staining in mice

[0439] The inventors next tested whether the enhanced membrane permeabilization and reduced aggregation tendency of antibodies in a CD5-containing buffer would increase the uniformity and depth of antibody staining throughout the mouse body. Different nervous systems (e.g., sympathetic and parasympathetic) regulate and coordinate organ function. To reveal the fine innervation of organs throughout the mouse body, the pan-neuronal marker protein gene product 9.5 (PGP9.5) was used to stain the peripheral neuronal network in adult pups (~4 weeks old, ~10 × 3 × 2 cm in size) ( Figure 1 e and Figure 4 a). After whole-body antibody labeling of mice using a CD5-containing buffer, the mice were made optically transparent and panoramically imaged using light-sheet microscopy.

[0440] The peripheral nervous system was uniformly stained, with a high level of staining in the vertebrae ( Figure 1 f) and adipose tissue ( Figure 1 g) There were no systematic differences in signal intensity between different tissues and at different depths in the mouse body.

[0441] For example, in the heart, the network of nerve fibers that runs through the ventricular myocardium is evident ( Figure 4 b). Splenic parenchyma shows a complex cone-shaped structure of nerve fibers ( Figure 4 c). The vagus nerve branches into smaller fiber bundles as it progresses toward the dorsal side of the spleen, where we also visualize the splenic nerve network. PGP9.5+ nerve fibers also innervate the hepatic sinusoids ( Figure 4 d) and are distributed along the hepatic duct from head to tail. In the gallbladder, a ganglion plexus consisting of a series of irregularly shaped ganglia ( Figure 4d) is clearly visible. In the small intestine, interconnected ganglion plexuses can be observed on the intestinal wall ( Figure 4 e).

[0442] In the 3D reconstruction of the scan, the nerves innervating different organs can be easily observed. For example, the vagus nerve can be traced, which provides parasympathetic innervation to the abdominal organs, connecting visceral organs such as the kidneys, adrenal glands, ureters, liver, spleen, and gastrointestinal (GI) tract ( Figure 1 h and Figure 4 f), Virtual reality visualization technology greatly facilitates this task. Compared with whole-organ antibody staining, whole-body tracking of mice can visualize the neural connections between different organs ( Figure 1 h). This will provide important clues to understanding the role of neural communication in normal physiology and disease.

[0443] Example 3 : Staining of lymphatic vessels and immune cells

[0444] To demonstrate the universality of this method, lymphatic vessels and immune cells were stained using lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and CD45, respectively.

[0445] At the whole mouse level, a finely structured lymphatic network can be observed throughout the body ( Figure 1 i). Details of lymphatic vessel organization in individual organs can be visualized. For example, LYVE-1+ vessels were observed in the hepatic sinusoidal endothelium and the superficial layer of the gastrocnemius muscle ( Figure 5 a and Figure 5 b). LYVE-1+ lymph nodes can be observed near the hind limbs. In particular, LYVE-1+ cells with different shapes can be seen in the adipose tissue ( Figure 5 c). The larger lymphatic vessels of the kidney branch into capillary lymphatic vessels with a tree-like structure ( Figure 5 d). Tracheal lymphatic vessels show a segmental pattern of interconnected vessels ( Figure 5 e). In the stomach, lymphatic vessels are unevenly distributed on the stomach wall and have tree-like branches ( Figure 5 f). Blunt-ended tubular lymphatic capillaries (lactamula) 20 The lymph nodes are clearly located in the intestinal villi, and a rich and orderly network of lymphatic plexuses and lymphatic vessels can be seen on the outer surface of the intestinal wall ( Figure 5 g-5i).

[0446] Previously, it was believed that the brain parenchyma lacked lymphatic vessels. 21,22 , although the central nervous system is lymphatically drained via meningeal lymphatics. Our whole-body immunolabeling data revealed that small, short lymphatic capillaries extend from the meninges into the brain parenchyma. Some LYVE-1+ lymphatic vessels were also observed connecting the olfactory bulb and cortex ( Figure 1j), which was observed by LYVE1 and PROX1 staining, respectively. We also found that lymphatic vessels entered the brain parenchyma surrounding the thalamus ( Figure 1 k), which was confirmed by LYVE1 and Podoplanin staining.

[0447] Example 4 : Studying the relationship between different physiological systems in the same mouse

[0448] The advantage of wildDISCO is that it is compatible with traditional validated labeling antibodies, allowing the relationship between different physiological systems to be studied in the same mouse. First, tyrosine hydroxylase (TH)+ sympathetic nerves and CD45+ immune cells were co-immunolabeled ( Figure 2 a-2c, Figure 6 Along parts of the vagus nerve, especially in the inferior mesenteric plexus ( Figure 2 a and Figure 2 b) Immune cells were found to be co-localized in large numbers, and there were frequent contacts between immune cells and sympathetic nerves in the intestinal wall ( Figure 2 c).

[0449] To better illustrate the neuro-immune interactions in the lymphatic system, especially in lymph nodes (LN), double staining of nerve fibers and lymphatic vessels was performed ( Figure 2 d, Figure 7-8 ). Among them, large LYVE1+ ( Figure 2 e) and PROX1+ (Prospero homeobox protein 1, a lymphatic endothelial marker) ( Figure 2 g ) LN ( Figure 2 e, Figure 2 g). CD45 staining confirmed that the observed structures were LN ( Figure 2 f ). Co-staining with the pan-neuronal marker PGP9.5 or the peripheral sympathetic neuron marker TH revealed the neuronal processes innervating LNs.

[0450] Next, wildDISCO was used to assess the impact of bioturbations. To this end, the gut-associated nervous system architecture of germ-free mice was compared with that of specific pathogen-free (SPF) standard mice. Double staining data of nerves and lymphatic vessels and nerves and immune cells have shown intricate details of the enteric nervous system in the entire gut ( Figure 2 a-2d). Studies on germ-free mice have found that the density of PGP9.5+ neural mesh networks is significantly reduced compared with wild-type mice. The density of the myenteric plexus is from 1.478 (×10 3 mm / mm 3 ) is reduced to 0.659 (×10 3 mm / mm 3 )( Figure 2h-2l, Fig. 9 ), confirming the importance of gut microbiota interactions for the development and / or maintenance of the mesenteric plexus 23 .

[0451] Example 5 :wildDISCO applied to cancer metastasis models

[0452] Tyrosine hydroxylase TH is co-stained with breast cancer cells MDA-MB-231. Cancer cells are clearly visible and tightly surrounded by TH+ nerves. Fig.10 shown.

[0453] Example 6 WildDISCO reveals the entire mouse arterial network

[0454] application α- smooth muscle muscle move Protein (α--SMA) was studied for its distribution in arteries at a systemic scale. Fig.11 shown.

[0455] Example 7 : Validation of other antibodies

[0456] To demonstrate the broad applicability of this method, the uniformity and depth of antibody staining were confirmed with other antibodies using the above-described method.

[0457] Tables 2 and 3 below show lists of validated antibodies.

[0458] Table 2. Validated antibodies

[0459]

[0460] Table 3. Other validated antibodies

[0461]

[0462] Example 8 : Comparison of different clearing methods for whole-body antibody staining in mice

[0463] like Fig.13 and Fig.14 As shown in the results in , wildDISCO showed superior staining in whole mice compared to other clearing techniques such as vDISCO, iDISCO, uDISCO, and PEGASOS methods.

[0464] Specifically, wildDISCO was evaluated against other established methods (vDISCO, iDISCO, uDISCO, and PEGASOS) for their effectiveness in staining whole mice using standard antibodies. In a direct side-by-side comparison using the synapsin 1 antibody, incomplete staining and blurred images of many organs (e.g., head and hind limbs) were observed from iDISCO-, uDISCO-, and PEGASOS-stained samples ( Fig.13 a, b). For example, wildDISCO enables uniform and deep penetration of antibodies throughout mouse internal organs, such as hind limbs, spinal cord, forelimbs, kidneys, and liver, compared with vDISCO ( Fig.13 b, c and Fig.14 ).

[0465] Materials and Methods: The detailed protocol of vDISCO has been described previously. 5 , where the previously used nanoenhancer was replaced by a commercial IgG antibody at a concentration of 25 µg per mouse, as shown in Table 4 below:

[0466] Table 4. vDISCO and wildDISCO protocols and buffers used

[0467]

[0468] For the iDISCO antibody staining method, we followed the original iDISCO+ publication 11 and the latest protocol updates at https: / / idisco.info. In brief, each step was adjusted to the whole body level rather than the organ level (e.g., methanol dehydration time was increased to 6 h). After pretreatment with methanol, the whole body was treated with a series of iDISCO+ solutions. For immunolabeling, the whole mouse was treated with the first solution (0.2% Tween-20, 5% DMSO, 3% donkey serum, 1 mg l -1 Heparin) in PBS (synapsin-1, CST, No. 5297, 25 µg in 250 ml, 1:10,000 dilution) for 7 days at 37°C, followed by a secondary solution (0.2% Tween-20, 5% DMSO, 3% donkey serum, 1 mgl -1 The secondary antibody (Alexa Fluor 647 goat anti-rabbit IgG antibody, Thermo Fisher Scientific, A-21245, 25 µg in 250 ml, 1:10,000 dilution) was incubated at 37°C for 7 days. Finally, the whole mouse was cleared until it became optically transparent.

[0469] For uDISCO and PEGASOS whole mouse labeling, immunostaining buffers and protocols were used as described for iDISCO. Subsequently, clearing steps for uDISCO and PEGASOS were based on the original publications. 41,35 conduct.

[0470] Example 9 :Visualization and analysis of tumor-associated TLS in tumor metastasis models

[0471] wildDISCO has the unique ability to study tertiary lymphoid structures (TLS) and their distribution in whole mice, such as Fig.15 The results are shown in .

[0472] Specifically, to visualize systemic cancer metastasis, 4T1 breast cancer cells were subcutaneously implanted into BALB / c mice as described in detail below. Fig.15 Conventional B cells, follicular dendritic cells, and T cell clusters and / or aggregates (representing TLS) were found in some metastatic sites, such as those in the lung and intestine ( Fig.15 b, il).

[0473] Materials and Methods: 4T1 breast cancer cells encoding EGFP and enhanced firefly luciferase were filtered through a 100 μm membrane and resuspended in RPMI 1640 medium (GIBCO, 11875093). For the subcutaneous injection model, 1 × 10 6 Cancer cells (50 μl) were injected into the fourth left and right mammary fat pads of 6-week-old female Balb / c mice. After 14 days, metastasis in mice was measured by bioluminescence using the IVIS Lumina II imaging system (Caliper Life Sciences). Briefly, mice were anesthetized with ketamine, fixed in an imaging chamber, and imaged 15 minutes after the injection of luciferin (150 mg kg-1, intraperitoneal injection). Bioluminescent signals were quantified using Living Image Software v.4.2 (Caliper Life Sciences). After metastasis was confirmed by IVIS imaging, mice were euthanized and stained with two antibodies, CD3 and CD23, to confirm TLS. CD5 (hepta(2,6-di-O-methyl)-β-cyclodextrin) was used in this experiment. Labeling solution (see the table above with wildDISCO buffer in Example 9). Clearing solution was THF, DCM, and BABB.

[0474] In summary, a new DISCO technology ("wildDISCO") is provided that allows high-resolution 3D imaging of the peripheral nervous system (PNS), lymphatic system, and vascular system throughout the mouse body. This technology can use conventional antibodies to stain mice whole-body and create a comprehensive biological atlas of the nervous, vascular, and lymphatic systems. It can reveal pathological changes, such as tertiary lymphatic structures in cancer, and can precisely track therapeutic molecules and cells, thereby enhancing our understanding of disease pathology and treatment.

[0475] Most diseases involve multiple interconnected physiological systems, but histological assessment of their pathology is currently limited to small tissue samples. Here we disclose wildDISCO, a technique involving cholesterol extraction that allows large fluorescent dye-containing markers (e.g., standard 150 kDa IgG antibodies) to penetrate deep tissues of chemically fixed whole mice. By combining wildDISCO with whole-body clearing of mice, a whole-body atlas of the nervous, immune, and lymphatic systems was generated and their intimate interactions in whole mice were shown.

[0476] For example, wildDISCO can reveal integrated neuronal, vascular, and lymphatic networks. By using this technique, PNS innervation in most organs can be observed, including the heart, lungs, liver, kidneys, stomach, and intestines. In addition, the vagus nerve innervating the gastrointestinal tract can be visualized. By using this technique, the heterogeneous infiltration of lymphatic capillaries in the center of the intestinal villi and the region-specific 3D villus lymphatic network can also be presented. Surprisingly, the lymph nodes were found to be innervated by a population of PNs with immunomodulatory potential. By using this technique, organ-specific vascular patterns and the cortical capillary network that serves as the main support for multiple bones can also be imaged. Therefore, mapping the entire mouse body system can provide a roadmap for a variety of studies, including neural circuits, immunomodulation, and angiogenesis throughout the mammalian body.

[0477] This invention allows unbiased imaging of transparent whole mouse bodies at cellular resolution, providing a comprehensive view of biological systems (nervous or lymphatic) in health and disease. WildDISCO does not rely on transgenic expression of fluorescent proteins, as it allows for uniform and simultaneous staining of structures throughout the mouse body using readily available IgG antibodies. The mouse head is a perfect example of the versatility of this method, as it combines both hard tissue (skull) and soft tissue (brain). Using wildDISCO, it is possible to map all lymphatic vessels in and around the brain parenchyma of an intact mouse head.

[0478] The wildDISCO technology enables uniform and simultaneous antibody staining throughout the mouse body. Previously unavailable 3D anatomical information becomes possible (e.g., with the help of VR visualization), allowing for a more complete understanding of the initiation, progression, and extent of pathology at the whole organism level.

[0479] Industrial Applicability

[0480] The methods and products of the present invention can be applied in industry, for example, they can be used to test biopharmaceutical drugs such as therapeutic antibodies.

[0481] References

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Claims

1. A method for preparing animal tissue for fluorescence microscopy, the method comprising: labeling a target molecule in a fixed animal tissue with a labeling solution, wherein the labeling solution comprises a labeling agent containing a fluorescent dye capable of binding to the target molecule, wherein the labeling agent has a molecular weight greater than 100 kDa, so as to obtain a fixed animal tissue labeled with the labeling agent containing the fluorescent dye, wherein the fixed animal tissue is treated with a permeabilization solution, and Wherein, the marking solution contains a cyclodextrin derivative.

2. The method according to claim 1, wherein the cyclodextrin derivative has a structure shown in the following formula: in: m is 6 to 8; R 2 , R 3 and R 6 are each independently selected from H and C1-C6 alkyl, the C1-C6 alkyl being optionally substituted with one or more groups selected from OH, oxo and COOH; and The degree of substitution (DS) refers to the number of non-hydrogen groups R in each pyranose unit. 2 , R 3 and R 6 The average number is 0.9 to 3, but when R 2 , R 3 and R 6 When each is selected from H and CH3, then DS ≥2.

3. The method according to claim 2, wherein m is 7.

4. The method according to claim 2 or 3, wherein: (a) R 2 and R 6 is a linear or branched C1-C6 alkyl group, and R 3 is H; or (b) R 2 and R 3 is a linear or branched C1-C6 alkyl group, and R 6 is H; or (c) R 3 and R 6 is a linear or branched C1-C6 alkyl group, and R 2 It's H.

5. The method according to any one of claims 2 to 4, wherein R 2 and R 6 is CH3, and R 3 It's H. 6 . The method according to claim 1 , wherein the cyclodextrin derivative is heptakis(2,6-di-O-methyl)-β-cyclodextrin.

7. The method according to any one of the preceding claims, wherein the fluorescent dye-containing labeling agent is an antibody conjugated to the fluorescent dye, the antibody being capable of binding to the target molecule.

8. The method of claim 7, wherein the antibody is IgG, IgA, IgM, IgD or IgE.

9. The method according to any one of the preceding claims, further comprising the following steps before labeling the target molecule in the fixed animal tissue with a labeling solution: contacting the fixed animal tissue with a first antibody capable of binding to a structure present in the fixed animal tissue, preferably a protein, a lipid, a DNA or an RNA, more preferably a protein present in the fixed animal tissue, The labeling agent containing a fluorescent dye can bind to the first antibody.

10. The method according to any one of the preceding steps further comprises: - a blocking step for blocking non-specific antigen binding of antibodies, wherein the blocking step is performed by treating the fixed animal tissue with a blocking solution prior to the labeling step, Wherein, the blocking solution contains animal serum.

11. A method according to any one of the preceding claims, comprising the following steps in order: - a decolorization, permeabilization and blocking step, which is performed by treating the fixed animal tissue with one or more solutions of heme removal, permeabilization and blocking; and - labeling the target molecule in the fixed animal tissue with a labeling solution, wherein the labeling solution comprises the cyclodextrin derivative and a labeling agent containing a fluorescent dye capable of binding to the target molecule, wherein the labeling agent has a molecular weight greater than 100 kDa, To obtain fixed animal tissue labeled with the labeling agent containing fluorescent dye.

12. A method according to any preceding claim, wherein the whole animal body is labelled.

13. Use of cyclodextrin derivatives for improving the labeling of target molecules in fixed animal tissues or whole animals with labeling agents containing fluorescent dyes, The cyclodextrin derivative is as defined in any one of claims 2 to 6.

14. The use according to claim 13, wherein: (a) R 2 and R 6 is a linear or branched C1-C6 alkyl group, and R 3 is H; or (b) R 2 and R 3 is a linear or branched C1-C6 alkyl group, and R 6 is H; or (c) R 3 and R 6 is a linear or branched C1-C6 alkyl group, and R 2 It's H.

15. The use according to claim 13 or 14, wherein R 2 and R 6 is CH3, and R 3 It's H.

16. The use according to any one of claims 13 to 15, wherein the cyclodextrin derivative is heptakis(2,6-di-O-methyl)-β-cyclodextrin.

17. The use according to any one of claims 13 to 16, wherein the molecular weight of the labeling agent containing the fluorescent dye is greater than 100 kDa and / or is an antibody conjugated with the fluorescent dye, and the antibody is capable of binding to the target molecule.

18. The use according to claim 17, wherein the antibody is IgG, IgA, IgM, IgD or IgE.

19. Use according to any one of claims 13 to 18, wherein the whole animal body is marked.

20. A composition comprising an antibody having a molecular weight greater than 100 kDa and a cyclodextrin derivative as defined in any one of claims 2 to 6, wherein the antibody is a labeling agent containing a fluorescent dye or a primary antibody.

21. The composition according to claim 20, wherein: (a) R 2 and R 6 is a linear or branched C1-C6 alkyl group, and R 3 is H; or (b) R 2 and R 3 is a linear or branched C1-C6 alkyl group, and R 6 is H; or (c) R 3 and R 6 is a linear or branched C1-C6 alkyl group, and R 2 It's H.

22. The composition according to claim 20 or 21, wherein R 2 and R 6 is CH3, and R 3 It's H.

23. The composition of any one of claims 20 to 22, wherein the cyclodextrin derivative is heptakis(2,6-di-O-methyl)-β-cyclodextrin.

24. A composition according to any one of claims 20 to 23 further comprising a zwitterionic surfactant.

25. A composition according to any one of claims 20 to 23 further comprising CHAPS and / or CHAPSO.

26. A composition according to claim 24 or 25, further comprising a nonionic surfactant.

27. The composition according to claim 24 or 25, further comprising Triton X-100 and / or IGEPAL CA-630.

28. A composition according to any one of claims 24 to 27, further comprising one or more, preferably all, of the following components in an aqueous buffer: - Animal serum; - organic solvents; and / or -Amino acids.

29. The composition according to any one of claims 24 to 27, further comprising the following components in phosphate buffered saline solution (PBS): - mammalian serum; - water-miscible organic solvents; and -Glycine.

30. A composition according to claim 28 or 29, wherein the components, if present, have the following concentrations: - Cyclodextrin derivatives: 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v; - Animal serum: 0.5 to 12, preferably 0.75 to 10, more preferably 1 to 3% v / v; - zwitterionic surfactant: 5 to 15, preferably 7.5 to 12.5, more preferably 10% w / v; - nonionic surfactant: 0.5 to 4, preferably 1 to 3, more preferably 2% w / v; - organic solvent: 5 to 20, preferably 7.5 to 15, more preferably 10% w / v; - Amino acids: 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v; And wherein the buffer concentration of the aqueous buffer is 0.05 to 0.2M, preferably 0.08 to 1.2M, more preferably 0.1M.

31. A composition according to any one of claims 20 to 30 having a pH of 7 to 7.4, preferably 7.2.

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