Methods for labeling, clearing, and imaging large tissues with antibodies

Abstract

The present invention relates to methods for labeling, clearing, and / or imaging large tissues using labeling agents such as antibodies, and related uses and products. The present invention includes, inter alia, 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 biopharmaceuticals, and analyzing the biodistribution of nanoparticles. Methods for preparing the animal tissues according to the present invention include whole-body labeling, clearing, and imaging methods. The methods of the present invention are advantageous in that they allow visualization of, for example, single cells within mammalian tissues, including whole mice or other large tissues, tumor metastasis at the single-cell level, and the distribution of biopharmaceuticals at the single-cell level in whole mice using labeling agents such as antibodies (e.g., the distribution of cancer-targeting therapeutic antibodies in whole animals, such as intact mice).

Description

[Technical Field]

[0001] The present invention relates to methods for labeling, clearing, and / or imaging large tissues using labeling agents such as antibodies, and related uses and products. The present invention includes, inter alia, 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 biopharmaceuticals, and analyzing the biodistribution of nanoparticles. Methods for preparing the animal tissues according to the present invention include whole-body labeling, clearing, and imaging methods. The methods of the present invention are advantageous in that they allow visualization of, for example, single cells within mammalian tissues, including whole mice or other large tissues, tumor metastasis at the single-cell level, and the distribution of biopharmaceuticals at the single-cell level in whole mice using labeling agents such as antibodies (e.g., the distribution of cancer-targeting therapeutic antibodies in whole animals, such as intact mice). [Background technology]

[0002] More than a century of specialized research has led to a detailed understanding of the overall anatomy of the human body and common model organisms, and detailed histological maps of many individual organs have been created. However, mapping the distribution, connectivity, and molecular composition of cell types throughout the entire body for a given experimental condition remains challenging. For example, although the nervous system is connected to every part of the mammalian body, there are no cellular-level maps of the nervous system to reveal the intricate relationships between organs and between organs and the central nervous system (Non-Patent Documents 1-3). Furthermore, most methods for imaging nerves or other cells in a whole-body context rely on transgenic animals (Non-Patent Documents 4, 5), which severely limits the flexibility of experimental design. Generating new transgenic animals to map changes in the distribution of associated proteins is usually extremely expensive and time-consuming, but such whole-body connectivity maps are necessary to understand the functional interdependencies between organ systems and how diseases that begin in one part of the body, such as during neurodegeneration or systemic inflammation, affect other parts. Whole-body imaging can capture cellular insights and provide integrated biological knowledge in healthy rodents. However, although mice are a commonly used animal model, they still lack fundamental information about their bodies, i.e., how the various organs and tissue systems are organized in the whole mouse.

[0003] Recent clearing methods have enabled the labeling and imaging of intact tissues (Non-Patent Document 6), mouse organs (Non-Patent Document 7), and bodies (Non-Patent Documents 3, 8-14), human organ masses (Non-Patent Document 15), and human embryos (Non-Patent Document 16). However, there are no widely applicable labeling methods suitable for the entire mouse body. Conventional whole-body imaging methods, such as CUBIC, PACT, and uDISCO, have enabled whole-body imaging, but they rely on transgenic expression of fluorescent proteins in a subset of cells, such as mice expressing Thy-1EGFP in neurons (Non-Patent Document 17). The vDISCO method (Non-Patent Document 5) uses small antibodies (one-tenth the size of IgG) called nanobodies to label the entire mouse body. In contrast to the thousands of conventional unconjugated antibodies developed over the past decades, very few nanobodies function in a histological setting.

[0004] Although homogeneous labeling of the whole body with small molecules (e.g., DNA-labeling dyes) or nanobodies can be achieved by cardiac pumping of the solution through the mouse vasculature (Non-Patent Document 5) (e.g., as described in Patent Document 1), this has proven difficult with standard IgG antibodies because 1) the antibodies degrade and / or precipitate during perfusion, 2) they do not homogeneously penetrate different tissue layers, including muscle and bone, and 3) cell membranes are not maximally permeabilized for deep antibody penetration into all tissues with diverse properties. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 224289 [Non-patent literature]

[0006] [Non-Patent Document 1] Richardson, DS et al. Tissue clearing. Nature Reviews Methods Primers 1, 84 (2021). [Non-patent document 2] Ueda, HR et al. Tissue clearing and its applications in neuroscience. Nature Reviews Neuroscience 21, 61-79 (2020). [Non-patent document 3] Chung, K. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332-337 (2013). [Non-patent document 4] Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56-62 (2007). [Non-patent document 5] Cai, R. et al. Panoptic imaging of transparent mice reveals whole-body neural projections and skull-meninges connections. Nature Neuroscience 22, 317-327 (2019). [Non-patent document 6] Rios, AC et al. Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging. Cancer Cell 35, 618-632.e616 (2019). [Non-Patent Document 7] Yang, B. et al. Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing. Cell 158, 945-958 (2014). [Non-patent document 8] Park, Y.-G. et al. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nature Biotechnology 37, 73-83 (2019). [Non-Patent Document 9] Ku, T. et al. Elasticizing tissues for reversible shape transformation and accelerated molecular labeling. Nature Methods 17, 609-613 (2020). [Non-Patent Document 10] Murray, E. et al. Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems. Cell 163, 1500-1514 (2015). [Non-Patent Document 11] Renier, N. et al. iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159, 896-910 (2014). [Non-Patent Document 12] Ertuerk, A. et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature Protocols 7, 1983-1995 (2012). [Non-Patent Document 13] Susaki, Etsuo A. et al. Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis. Cell 157, 726-739 (2014). [[ID=I8]] [Non-Patent Document 14] Dodt, H.-U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nature Methods 4, 331-336 (2007). [Non-Patent Document 15] Zhao, S. et al. Cellular and Molecular Probing of Intact Human Organs. Cell 180, 796-812.e719 (2020). [Non-Patent Document 16] Belle, M. et al. Tridimensional Visualization and Analysis of Early Human Development. Cell 169, 161- 173.e112 (2017). [Non-Patent Document 17] Feng, G. et al. Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP. Neuron 28, 41-51 (2000). Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, further improved and more versatile methods for the preparation and analysis of tissues, including whole animal and large mammalian brains, are needed. Specifically, indirect immunolabeling of whole mice with conventional primary and secondary antibodies would be a particularly valuable method for many biological applications, such as whole-body mapping of cells of interest. [Means for solving the problem]

[0008] Here, we provide a new technique capable of high-resolution 3D imaging of the peripheral nervous system (PNS), lymphatic system, and vasculature throughout the entire body of an animal (e.g., a mouse). The underlying technique, called wildDISCO (wild-type mouse immunolabeling and DISCO ablation), is a chemical method that enhances the penetration of standard labeling agents, such as antibodies (preferably greater than 100 kDa in size, e.g., approximately 150 kDa), throughout the entire animal body (e.g., approximately 2 cm thick in a mouse body). This method may involve cholesterol extraction for permeabilization to ensure uniform penetration and staining across tissues throughout the mouse body, including, for example, muscle, bone, brain, and spinal cord. Combining whole-body antibody labeling with DISCO-based tissue clearing provides a body-wide map of cell type and protein distribution with unprecedented ease, further advancing our understanding of biological systems. wildDISCO can reveal integrated neural, vascular, and lymphatic networks. Using this technique, we can observe PNS innervation in most organs, including the heart, lungs, liver, kidneys, stomach, and intestine. Furthermore, we can visualize the vagus nerve, which innervates the gastrointestinal tract. Using this technique, we can also reveal the heterogeneous lymphatic vessels that permeate the center of the intestinal villi and the region-specific 3D villous lymphatic network. Surprisingly, we found that lymph nodes are innervated by a population of PNs with immunoregulatory functions. Using this technique, we can also image organ-specific vascular patterns and transcortical capillary networks that serve as the primary support for multiple bones. Therefore, mapping the mouse whole-body system provides a roadmap for diverse studies, including neural circuits, immune regulation, and angiogenesis in the mammalian body.

[0009] Furthermore, the present invention enables unbiased imaging of transparent whole-body mice at cellular resolution, providing a comprehensive picture of biological systems (neural or lymphatic) in health and disease. wildDISCO enables uniform and simultaneous staining of whole-body mouse structures using off-the-shelf IgG antibodies, without relying on transgenic expression of fluorescent proteins. The present invention provides a versatile method. The mouse head, a combination of hard (skull) and soft (brain) tissues, is a perfect example of the versatility of the method. wildDISCO can be used to map lymphatic vessels in and around the brain parenchyma of an intact mouse head. In summary, the wildDISCO technology achieves uniform and simultaneous antibody staining throughout large tissues, such as the entire body of a mouse. Previously unavailable 3D anatomical information (e.g., aided by VR visualization) may provide a more comprehensive understanding of the initiation, progression, and extent of pathology at the whole-organism level in mice.

[0010] The inventors reasoned that optical imaging of the present invention, including the transparent tissue mouse, could be useful as a powerful preclinical approach for detecting fluorescently labeled cancer cells and / or therapeutic antibodies at cellular resolution within the intact body. Fluorescent labeling of cancer cells in vitro or in vivo is typically achieved through the endogenous expression of fluorescent proteins, such as GFP, YFP, and mCherry, which emit light in the visible spectrum. However, many tissues within the body also exhibit high autofluorescence in this range (Tuchin, 2016; Zipfel et al., 2003), which precludes reliable detection of single cancer cells through the several-centimeter thickness of the intact mouse body.

[0011] In a preferred embodiment of the present invention, labeling cells, such as cancer cells, with antibodies tagged with fluorescent dyes that have emission peaks in the far-red range is advantageous for overcoming the autofluorescence signal by providing a higher signal-to-background ratio for reliable detection of single cells. Toward this goal, the present inventors have developed an improved method for preparing animal tissues for fluorescence microscopy. Preferably, the method does not rely on endogenously expressed fluorescent proteins and instead uses antibody-based whole-body labeling (e.g., immunolabeling) techniques to specifically label endogenous cellular proteins with fluorescent dyes, such as Alexa and Atto dyes, preferably in the far-red spectrum. Organic solvent-based clearing methods, such as whole-body DISCO clearing (see Pan et al., 2016, incorporated by reference in its entirety for all purposes), can be included in the methods of the present invention. The methods of the present invention are advantageous in that they allow visualization of cells, such as cancer cells, in intact see-through mice, even in highly autofluorescent tissues.

[0012] The methods of the present invention can be used, for example, to evaluate tumor metastasis and the biodistribution of cancer cell-targeting antibodies in mice. This discovery can be demonstrated, for example, using mice implanted with human breast cancer cells and injected with the therapeutic monoclonal antibody 6A10 against carbonic anhydrase XII (Cα12) (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, monitor tumor drug-target interactions at the single-cell level in intact mice, and further determine the phenotype of a defined tumor microenvironment through rehydration of removed tissue and subsequent antibody labeling.

[0013] The present method can be used, for example, to analyze micrometastases and therapeutic anti-tumor antibody distribution in tissues, such as whole mice, at cellular resolution. The present method is unbiased because it can label and detect target molecules in animal tissues at single-cell resolution without dissecting the animal tissue (e.g., whole mice) before analyzing it. Advantageously, the animal tissues that can be prepared and analyzed at single-cell resolution by the present invention without prior dissection are larger than those by previously known methods. Therefore, the present method minimizes bias introduced by dissection of tissue (and subsequent separate analysis of different dissected portions of the animal tissue). For example, bias that can be introduced by analyzing only a given organ or portion of that organ can be minimized by the present method. In a non-limiting embodiment, the organic solvent used by the present method can shrink the animal tissue to a smaller size, making it more accessible to fluorescence microscopy using a microscope objective at its given maximum working distance, contributing to this advantageous effect. The method of the present invention is also advantageous over previous methods in that it allows removal of tissues including skin, for example, whole adult mice including their skin.

[0014] The method of the present invention is also advantageous in that it can be easily applied in a variety of laboratories without highly specialized equipment, since even imaging with commonly used epifluorescence microscopes can detect more detail in the intact see-through mouse than can be visualized through bioluminescence imaging. The methods of the present invention can also reduce the time and cost required to investigate tumor micrometastases at the cellular level, for example, in the whole body of a mouse. Furthermore, because researchers can easily evaluate the whole body of a mouse instead of selected tissues / organs and because the methods are highly sensitive (single cells can be identified and quantified throughout the body), the number of mice used in a study can also be significantly reduced using the methods of the present invention. Therefore, the methods of the present invention presented herein can facilitate the translation of new therapies into the clinic much more efficiently than conventional methods. Furthermore, unlike known tissue clearing methods, such as the CUBIC and PACT methods, which weaken the tissue, the method for preparing animal tissue for fluorescence microscopy according to the present invention stiffens the animal tissue. Therefore, advantageously, the animal tissue obtainable by the method of the present invention is suitable for dissection into different sections and further analysis of the sections after dissection by fluorescence microscopy. It is understood that, according to the present invention, dissection of the animal tissue obtainable by the method of the present invention may not be necessary, since the animal tissue that can be prepared and analyzed at single-cell resolution by the present invention without prior dissection is larger than with previously known methods. However, when dissection is desired, the 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.

[0015] Tissue labeling such as whole-body immunostaining using antibodies Imaging endogenous proteins, such as endogenous fluorescent proteins, in thick biological tissues poses significant challenges, including autofluorescence in the blue-green spectrum and bleaching during long imaging and storage. In an exemplary embodiment of the present invention, to achieve high signal quality (e.g., for single tumor cell detection in whole adult mice), a primary antibody (binding to an endogenous protein, e.g., an endogenous protein of a cancer cell) can be labeled with a secondary antibody as a labeling agent, such as a secondary antibody conjugated to a fluorescent dye, e.g., Atto or Alexa dye. This approach is advantageous in that it enhances the signal-to-background ratio and allows visualization of single cells in tissues, especially within centimeter-thick mouse bodies. It will be appreciated that the present invention can further improve imaging quality by using fluorescent dyes in the far-red or longer wavelength spectrum, such as near-infrared fluorescent dyes, potentially enabling the study of intracellular structures / molecules in whole mouse tissues (for examples of suitable fluorescent dyes, see Hong et al., 2017, incorporated herein by reference in its entirety for all purposes).

[0016] The present invention employs labeling with a fluorescent dye-containing labeling agent (e.g., an antibody conjugated to a fluorescent dye) that preferably has a molecular weight of more than 100 kDa, e.g., 110 kDa or more, 120 kDa or more, 130 kDa or more, or 140 kDa or more. Antibodies that can be conjugated to a fluorescent dye and used in the present invention include, but are not limited to, IgG molecules (e.g., IgG1, IgG2, IgG3, or IgG4), IgD molecules, IgE molecules, IgA molecules, and IgM molecules.

[0017] The term "antibody" as used herein is described in Chapter 7 of Paul, WE (Ed.): Fundamental Immunology 2nd Ed. Raven Press, Ltd., New York 1989. Without limitation, the term "antibody" encompasses antibodies derived from any suitable source species, including chicken, and mammals such as mouse, goat, non-human primate, and human. Antibodies may be monoclonal or polyclonal. Such antibodies may be prepared by methods well known in the art. The term "antibody" also encompasses, but is not limited to, isolated and modified antibodies, e.g., genetically engineered antibodies, e.g., chimeric, humanized, or human antibodies. Preferred antibodies are listed in Tables 2 and 3. In one embodiment, new antibodies can be generated for the methods and uses of the invention to study pathologies that affect the entire body. For example, labeling agents (e.g., antibodies) that can be used as inflammation or infection markers would be useful for gathering unbiased readouts in whole mice for inflammatory disorders, such as multiple sclerosis or rheumatoid arthritis, or infectious diseases that affect the entire body.

[0018] Detection of Micrometastases According to the Invention For example, unbiased, high-throughput mapping of tumor micrometastases at cellular resolution throughout the entire body of a rodent could be a valuable tool for elucidating the biology behind tumor cell dissemination. In an exemplary embodiment, the present invention encompasses the wildDISCO method, which can be used for volumetric imaging of tumor micrometastases throughout the entire body of a mouse. While the use of a single-plane laser scanning light-sheet microscope is the most preferred embodiment of the analytical method according to the present invention, for example, to detect cancer cells in a see-through mouse, even the use of a standard fluorescence microscope can provide novel insights. Furthermore, epifluorescence imaging is useful for determining regions of interest by performing a simple scan of the cleared mouse body within minutes before collecting a large dataset using light-sheet microscopy. Subsequent light-sheet microscopy imaging can be focused only on organs / regions of interest based on the epifluorescence data. This approach significantly accelerates the research being conducted and reduces the amount of data being analyzed. Advantageously, the method of the present invention can be adapted to detect and map cancer metastases in the whole mouse body at the cellular level, and can identify the precise location of single disseminated cancer cells. The method of the present invention allows for re-probing of identified metastatic tissues with conventional antibodies, for example, gene expression profiling via RNA-seq and proteomics (by mass spectrometry). 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. Thus, the method of the present invention allows for characterization and molecular screening of single tumor cells identified in micrometastases and distant organs. According to the present invention, molecular markers of specific subtypes of tumor cells, such as cancer stem cells, or of inflammatory cells and extracellular matrix components from the tumor microenvironment, such as cancer-associated fibroblasts, T cells, and macrophages, can be used to help determine their precise spatiotemporal distribution in metastatic tissues, e.g., the whole body of a rodent.

[0019] Analysis of biodistribution of biopharmaceuticals according to the present invention Accurate assessment of the biodistribution of biopharmaceuticals (e.g., antibody drugs) is important for evaluating their specificity and usefulness for treatments such as tumor therapy, but no method exists that can provide this information at the cellular level in intact organisms. The method of the present invention (also referred to in exemplary embodiments as the "wildDISCO" method) is a novel tool that can be used to study not only the distribution of single tumor cells but also the distribution of antibody-based therapies. The method of the present invention can identify antibody-targeted tumor cells, particularly in metastases within various organs, including the lung, kidney, brain, and liver. The analytical methods of the present invention can also be advantageous in that they can be used to detect binding of biopharmaceuticals (e.g., therapeutic antibodies) to non-target tissues (e.g., non-cancerous tissues in the case of cancer treatment antibodies) to indicate potential off-target effects.

[0020] Analysis of biodistribution of nanoparticles according to the present invention In an exemplary method for analyzing nanoparticle biodistribution, nanoparticles (DNA origami or carbon nanotubes) can be conjugated to polymers such as PEG to enhance circulation time and stability. They can also be tagged with moieties such as antibodies, peptides, or aptamers for targeting. For example, CpG peptides can be used to target immune cells. Finally, they can also be conjugated to fluorescent dyes (e.g., Alexa or Atto dyes) for use in the methods of the present invention. Conjugated nanoparticles can be dissolved in PBS at concentrations ranging from 200 nM to 2 μM. 100–200 μL of this solution is then injected into mice either intravenously or intravenously. The mice are then perfused as quickly as possible for 3 hours (or longer). The biodistribution of nanoparticles is assessed using the methods of the present invention. Thus, the present invention provides an advantageous labeling and analysis platform that can visualize and analyze, for example, tumor micrometastases and antibody-based therapies at single-cell resolution in the whole body of a mouse. The methods of the present invention are time- and cost-efficient and can be used to investigate a variety of biomedical problems, for example, those related to various pathologies or developmental processes that affect the entire organism. Thus, the present invention encompasses the following preferred embodiments.

[0021] Embodiment 1. A method for preparing animal tissue for fluorescence microscopy, said method comprising: a) optionally decalcifying fixed animal tissue with a decalcifying solution; b) optionally decolorizing fixed animal tissue with a solution to remove heme; c) labeling a target molecule in the fixed animal tissue with a labeling solution containing a fluorescent dye-containing labeling agent capable of binding to the target molecule to obtain a fixed animal tissue labeled with the fluorescent dye-containing labeling agent, wherein the molecular weight of the labeling agent is greater than 100 kDa; wherein the fixed animal tissue is preferably treated with a permeabilization solution before and / or during the labeling of the fixed animal tissue in step c), Preferably, the permeabilization solution and the labeling solution are the same or different solutions, and wherein: the permeabilization solution and / or the labeling solution comprises a cyclodextrin derivative; d) optionally clearing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent with a clearing solution comprising an organic solvent to obtain the animal tissue for fluorescence microscopy.

[0022] 2. Cyclodextrin derivatives have the following chemical formula:

[0023] [ka] (In the formula, m is 6-8; R 2 , R 3 and R 6 are each independently selected from H and optionally substituted alkyl; and Non-hydrogen group R per glucopyranose unit 2 , R3 and R 6 The method according to embodiment 1, wherein the structure has a degree of substitution (DS), which represents the average number of 3. The method of embodiment 2 above, wherein the optionally substituted alkyl is a straight-chain or branched C1-C6 alkyl optionally substituted with one or more groups selected from OH, SO3H, SO3Na, oxo, and COOH. 4.R 2 , R 3 and R 6 are each independently selected from H and linear or branched C1-C4 alkyl optionally substituted with one or more groups selected from OH, SO3H, SO3Na, oxo, and COOH. 5. The method of any one of embodiments 2 to 4, wherein DS≧0.4. 6. The method of any one of embodiments 2 to 5, wherein DS≧0.5. 7. The method of any one of embodiments 2 to 6, wherein DS≧0.6. 8. The method of any one of embodiments 2 to 7, wherein DS≧0.7. 9. The method of any one of embodiments 2 to 8, wherein DS≧0.8. 10. The method of any one of embodiments 2 to 9, wherein DS≧0.9. 11. A method according to any one of embodiments 2 to 10, wherein DS≧1.0. 12. A method according to any one of embodiments 2 to 11, wherein DS≧1.5. 13. A method according to any one of embodiments 2 to 12, wherein DS≧1.8. 14. A method according to any one of embodiments 2 to 13, wherein DS≧1.9. 15. A method according to any one of embodiments 2 to 14, wherein DS≧2.0. 16. A method according to any one of embodiments 2 to 15, wherein DS≧2.5. 17. The method of any one of embodiments 2 to 16, wherein DS≦3.0. 18. The method of any one of embodiments 2 to 17, wherein DS≦2.8. 19.R 2 , R 3 and R 6 are each independently selected from H and C1-C6 alkyl optionally substituted with one or more oxo and / or OH. 20. The use according to any one of embodiments 1 to 19, wherein the cyclodextrin derivative is not methyl-β-cyclodextrin with a degree of substitution of 1.8.

[0024] 21. The use according to any one of embodiments 1 to 20, wherein the cyclodextrin derivative is not a methyl-β-cyclodextrin with a degree of substitution <2.0. 22.R 2 , R 3 and R 6 22. The method of any one of embodiments 2-21, wherein DS≧2 when each is selected from H and CH3. 23. A method according to any one of embodiments 2 to 22, wherein the DS is 1.8 to 2.0. 24.R 2 , R 3 and R 6 is each independently selected from H and CH3. 25.R 2 , R 3 and R 6 is each independently selected from H and CH3. 26. The method of embodiment 25, wherein the DS is 1.8. 27.(a)R 2 and R 6 is a linear or branched C1-C6 alkyl, and R 3 is H, or (b)R 2 and R 3 is a linear or branched C1-C6 alkyl, and R 6 is H, or (c)R 3 and R6 is a linear or branched C1-C6 alkyl, and R 2 24. The method of any one of embodiments 2-23, wherein 28.R 2 and R 6 is CH3 and R 3 27. The method of any one of embodiments 2-26, wherein 29.R 2 , R 3 and R 6 are each independently H and C(O)C 1-5 27. The method of any one of embodiments 2 to 26, wherein the alkyl is selected from alkyl. 30.R 2 , R 3 and R 6 are each independently selected from H and C(O)CH.

[0025] 31. A method according to any one of embodiments 2 to 30, wherein the DS is 2.5 to 3.0. 32.R 2 , R 3 and R 6 is each C(O)CH3. 33.R 2 , R 3 and R 6 are each independently H, -C 1-2 Alkyl(OH)C 1-3 Alkyl and -C 1-2 24. The method of any one of embodiments 2 to 23, wherein the alkyl group is selected from alkyl-OH. 34. The method of any one of embodiments 2-33, wherein when alkyl is substituted by one or more OH groups, the alkyl is a linear or branched C3-C6 alkyl, and / or has a DS≧0.9. 35. A method according to any one of embodiments 2 to 34, wherein the DS is 0.8 to 3.0. 36. A method according to any one of embodiments 2 to 35, wherein the DS is 0.9 to 3.0. 37.R 2 , R 3 and R6 The method according to any one of Embodiments 2 to 36, wherein each is independently selected from H and -CH2CH(OH)CH3. 38. The method according to any one of Embodiments 2 to 37, wherein DS is 0.9. 39. R 2 R 3 and R 6 The method according to any one of Embodiments 2 to 38, wherein each is independently selected from H and -CH2CH2OH. 40. The method according to Embodiment 39, wherein DS is 0.8.

[0026] 41. R 2 R 3 and R 6 The method according to any one of Embodiments 2 to 40, wherein each is independently selected from H and a linear or branched C1 - C5 alkyl optionally substituted by COOH. <00​​​​​​​​​​​​​​​​​​​​​​​​​​48. The method of any one of the previous embodiments, wherein the cyclodextrin derivative is selected from (2-hydroxypropyl)-β-cyclodextrin, triacetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, γ-cyclodextrin and α-cyclodextrin; more preferably (2-hydroxypropyl)-β-cyclodextrin or heptakis(2,6-di-O-methyl)-β-cyclodextrin; most preferably heptakis(2,6-di-O-methyl)-β-cyclodextrin. 49. The method of any one of the preceding embodiments, further comprising blocking to block non-specific antigen binding of antibodies, wherein the blocking is performed by treating the fixed animal tissue with a blocking solution prior to labeling. 50. The method of embodiment 49, wherein the blocking solution comprises animal serum.

[0027] 51. The method according to embodiment 50, wherein the animal serum is mammalian serum, preferably goat serum or donkey serum, more preferably goat serum. 52. The method of any one of embodiments 49-51, wherein the blocking solution further comprises a surfactant. 53. The method according to embodiment 53, wherein the surfactant is a non-ionic surfactant, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100. 54. Blocking solution - Animal serum at a concentration of 1 to 15, preferably 3 to 10, more preferably 3% v / v, and / or - a non-ionic surfactant at a concentration of 0.5 to 4, preferably 1 to 3, more preferably 2% w / v in an aqueous buffer, preferably phosphate buffered saline (PBS), optionally having a buffer concentration of 0.05-0.2 M, preferably 0.08-1.2 M, more preferably 0.1 M. 55. The method of any one of the preceding embodiments, wherein treating with the permeabilization solution is performed simultaneously with treating with the blocking solution. 56. The method of any one of the previous embodiments, wherein the permeabilization solution and the blocking solution are the same solution. 57. The method of any one of the preceding embodiments, comprising a). 58. A method according to any one of the preceding embodiments, wherein in a) the decalcification solution is selected from a solution containing EDTA and NaHCO3, a solution containing formic acid, a solution containing HNO3, or a solution containing HCl, and preferably the pH value of the decalcification solution is 8-9. 59. The method of any one of the previous 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. 60. The method of any one of embodiments 57 to 59, wherein the blocking as specified in any one of embodiments 49 to 56 is carried out after a).

[0028] 61. The method of any one of the preceding embodiments, comprising b). 62. The method of any one of the preceding embodiments, wherein b) is performed by perfusing the fixed animal tissue with the solution for heme removal, and / or the solution for heme removal is a heme chelating solution. 63. The method according to any one of the preceding embodiments, wherein in b), the solution for heme removal comprises an amino alcohol suitable for heme removal and optionally a surfactant. 64. The method of embodiment 63, wherein the solution for removing heme comprises a detergent, and the detergent is an ionic detergent, a non-ionic detergent, a zwitterionic detergent, a chaotropic detergent, or a combination thereof. 65. The method of embodiment 64, wherein the surfactant is an ionic surfactant that is sodium dodecyl sulfate or sodium deoxycholate. 66. The method of embodiment 64, wherein the surfactant is a non-ionic surfactant that is 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether, or polyoxyethylene(20) sorbitan monolaurate. 67. The method of embodiment 64, wherein the surfactant is a zwitterionic surfactant that is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate. 68. The method of embodiment 64, wherein the surfactant is a chaotropic surfactant that is urea. 69. The method of any one of embodiments 63 to 68, wherein the amino alcohol is N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, N-butyldiethanolamine, N-methyldiethanolamine, 4-(2-hydroxyethyl)morpholine, N-ethyldiethanolamine, 2-(diisopropylamino)ethanol, 4-methylmorpholine N-oxide, or 1-(2-hydroxyethyl)piperidine. 70. The method of any one of embodiments 63-69, wherein the amino alcohol is N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine.

[0029] 71. The method of embodiment 70, wherein the solution for removing heme is a 1:2 or 1:3 dilution of the following reagents, preferably 0.1 M PBS; 25 wt% urea, 25 wt% N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, 15 wt% Triton X-100 in 0.1 M PBS. 72. The method of any one of the previous embodiments, wherein in step b), the solution for removing heme comprises an oxidizing agent for oxidizing heme. 73. The method of embodiment 72, wherein the oxidizing agent for the oxidation of heme is benzyl peroxide, 3-chloroperoxybenzoic acid, or magnesium monoperoxyphthalate hexahydrate. 74. The method of embodiment 72, wherein the oxidizing agent for the oxidation of heme is benzyl peroxide. 75. The method of any one of the previous embodiments, wherein step b) is performed simultaneously with step c). 76. The method of any one of the previous embodiments, wherein the solution for removing heme is the same solution as the permeabilization solution and / or labeling solution. 77. The method of any one of the preceding embodiments, wherein the permeabilization solution and / or the labeling solution comprises a surfactant, and the surfactant is an ionic surfactant, a non-ionic surfactant, a zwitterionic surfactant, a chaotropic surfactant, or a combination thereof. 78. The method of any one of the preceding embodiments, wherein the permeabilization solution and / or the labeling solution comprises a non-ionic detergent and / or a zwitterionic detergent. 79. The permeabilization solution and / or the labeling solution are a non-ionic surfactant, preferably selected from Triton X-100 and IGEPAL CA-630, more preferably Triton X-100; and Optionally, the zwitterionic detergent is selected from CHAPS or CHAPSO, more preferably CHAPS. 80. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 110 kDa or greater.

[0030] 81. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 120 kDa or greater. 82. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 130 kDa or greater. 83. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 140 kDa or greater. 84. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150 kDa or greater. 85. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 900 kDa or less. 86. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 500 kDa or less. 87. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 385 kDa or less. 88. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 300 kDa or less. 89. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 200 kDa or less. 90. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 180 kDa or less.

[0031] 91. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150 kDa or less. 92. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 110-900 kDa. 93. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 120-500 kDa. 94. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 130 to 385 kDa. 95. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 140-300 kDa. 96. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150-200 kDa. 97. The method of any one of the preceding embodiments, wherein the molecular weight of the fluorescent dye-containing labeling agent is 150-180 kDa. 98. The method of any one of the preceding embodiments, wherein the fluorescent dye is capable of emitting infrared or red fluorescence. 99. The method of any one of the preceding embodiments, wherein the fluorescent dye is capable of emitting near-infrared or far-infrared fluorescence. 100. The method of any one of the preceding embodiments, wherein the wavelength of the emission maximum of the fluorescent dye is greater than 480 nm.

[0032] 101. The method of any one of embodiments 1 to 100, wherein the wavelength of the emission maximum of the fluorescent dye is greater than 500 nm. 102. The method of any one of embodiments 1 to 101, wherein the wavelength of the emission maximum of the fluorescent dye is higher than 550 nm. 103. The method of any one of embodiments 1 to 102, wherein the wavelength of the emission maximum of the fluorescent dye is higher than 590 nm. 104. The method of any one of embodiments 1 to 103, wherein the wavelength of the emission maximum of the fluorescent dye is greater than 600 nm. 105. The method of any one of embodiments 1 to 104, wherein the wavelength of the emission maximum of the fluorescent dye is higher than 640 nm. 106. The method of any one of embodiments 1 to 105, wherein the wavelength of the emission maximum of the fluorescent dye is greater than 700 nm. 107. The method of any one of embodiments 1 to 106, wherein the wavelength of the emission maximum of the fluorescent dye is in the range of 640 nm to 700 nm. 108. The method of any one of embodiments 1 to 107, wherein the wavelength of the emission maximum of the fluorescent dye is less than 1000 nm or less than 900 nm. 109. The method of any one of embodiments 1 to 108, wherein the wavelength of the emission maximum of the fluorescent dye is less than 800 nm. 110. The method of 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, preferably selected from those listed in Tables 2 and 3.

[0033] 111. The method of embodiment 110, wherein the antibody is IgG, IgA, IgM, IgD or IgE. 111. The method of embodiment 110 or 111, wherein the antibody is an IgG. 112. The method of embodiment 111, wherein the antibody is IgG1. 113. The method of embodiment 111, wherein the antibody is IgG2. 114. The method of embodiment 111, wherein the antibody is IgG3. 115. The method of embodiment 111, wherein the antibody is IgG4. 116. The method of any one of the preceding embodiments, wherein step c) is carried out by perfusing fixed animal tissue with the labeling solution containing the fluorescent dye-containing labeling agent. 117. The method of any one of the preceding embodiments, wherein the fluorescent dye-containing labeling agent is a fluorescent dye, and the fluorescent dye is capable of binding to the target molecule. 118. The method of any one of the preceding embodiments, wherein the fluorescent dye-containing labeling agent comprises a fluorescent dye, preferably, the fluorescent dye is Nissl, propidium iodide, methoxy-xO4, cresyl violet acetate, pyronin Y, thiazine red, lectin, DiI, Atto dye, Alexa Fluor dye, Cy dye, and To-pro3. 119. The method of any one 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, ATTO550, ATTO647, Cy7, Cy5, and Cy3. 120. The method of any one of the previous embodiments, wherein the refractive index of the organic solvent deviates from the refractive index of the animal tissue by no more than 5%.

[0034] 121. The method of any one of the preceding embodiments, wherein the refractive index of the clearing solution comprising an organic solvent deviates from the refractive index of the animal tissue by no more than 2%. 122. The method according to any one of the previous embodiments, wherein the refractive index of the clarifying solution containing an organic solvent is 1.500 to 1.600. 123. The method according to any one of the previous embodiments, wherein the refractive index of the clarifying solution containing an organic solvent is 1.520 to 1.580. 124. The method of any one of the previous embodiments, wherein the organic solvent comprises benzyl alcohol, benzyl benzoate, dibenzyl ether, ethyl 3-phenyl-2-propenoate, allyl 3-phenylacrylate, PEG (Mn=200-1000), PEGDA (Mn=200-1000), PEGMA (Mn=200-1000), 1-phenylnaphthalene, and / or diphenyl ether. 125. The method of any one of the previous embodiments, wherein the clarifying solution comprising an organic solvent further comprises an antioxidant. 126. The method of any one of the preceding embodiments, wherein the organic solvent-containing clarifying solution consists of benzyl alcohol, benzyl benzoate, and diphenyl ether, and an antioxidant, in a volume ratio of 4:8:3 to 10:20:3. 127. The method of embodiment 125 or 126, wherein the antioxidant is DL-α-tocopherol. 128. The method of any one of embodiments 125-127, wherein the antioxidant is present in the clarifying solution in an amount of 0.4% by volume. 129. The method of any one of the preceding embodiments, wherein step d) is carried out by perfusing the fixed animal tissue labeled with the fluorescent dye-containing labeling agent with a clearing solution comprising the organic solvent. 130. The method of embodiment 129, wherein the fixed animal tissue labeled with the fluorescent dye-containing labeling agent is perfused with a clearing solution comprising the organic solvent for at least 6 hours.

[0035] 131. The method according to embodiment 129 or 130, wherein step d) further comprises perfusing with an increasing gradient of a dehydrating solution containing 0 to 100% by volume of an additional organic solvent before perfusing with the clearing solution. 132. The method of embodiment 131, wherein the membrane is perfused with an increasing gradient of a dehydration solution comprising 0-100% by volume of an additional organic solvent, followed by a delipidation solution comprising an additional organic solvent. 133. The method of embodiment 132, wherein the further organic solvent is tert-butanol, tetrahydrofuran (THF), methanol, ethanol, or 1,4-dioxane, and the increasing gradient perfusion is carried out at a temperature above the melting temperature of said further organic solvent. 134. The method of embodiment 132 or 133, wherein the further organic solvent is dichloromethane, chloroform, methanol, hexane, butanol, ethyl acetate, tert-butyl methyl ether, and the perfusion with said further organic solvent is carried out at a temperature above the melting temperature of said further organic solvent. 135. The method of any one of the preceding embodiments, wherein the labeling solution, the permeabilization solution, and the clearing solution are actively delivered by pressure, preferably by pressure with a pump. 136. A method according to any one of the preceding embodiments, wherein the labeling of the target molecule with the labeling solution and the treatment with the permeabilization solution are carried out by perfusion at a pressure higher than 80 mmHg, preferably higher than 150 mmHg. 137. A method according to any one of the preceding embodiments, wherein the labeling of the target molecule with the labeling solution and the treatment with the permeabilization solution are carried out by perfusion at a pressure of 220 to 240 mmHg, preferably at a pressure of 230 mmHg. 138. The method of any one of the preceding embodiments, wherein the fixed and destained animal tissue is treated with the permeabilization solution prior to the labeling of the target molecule in step c), and the permeabilization solution and the labeling solution are different solutions. 139. The method of embodiment 138, wherein the permeabilization solution is a dehydrated solution as specified in embodiment 132 or 133. 140. The method according to embodiment 138, wherein the permeabilization solution is a delipidation solution as specified in embodiment 132 or 133.

[0036] 141. The method of embodiment 138, wherein the permeabilization solution comprises acetic acid. 142. The method of embodiment 138, wherein the permeabilization solution comprises guanidine hydrochloride and / or sodium acetate. 143. The method of any one of embodiments 1 to 137, wherein the fixed animal tissue is treated with the permeabilization solution during the labeling of the target molecule in step c), and the permeabilization solution and the labeling solution are the same solution. 144. The method according to any one of the previous embodiments, wherein step b) is carried out by perfusing the fixed animal tissue with said solution for removal of heme at a pressure higher than 80 mmHg, preferably higher than 150 mmHg. 145. The method according to any one of the previous embodiments, wherein step b) is carried out by perfusing the fixed animal tissue with said solution for removal of heme at a pressure of 220-240 mmHg, preferably 230 mmHg. 146. The method according to any one of embodiments 130 to 145, wherein the perfusion in step d) is carried out at a pressure higher than 80 mmHg, preferably higher than 150 mmHg. 147. The method according to any one of embodiments 130 to 145, wherein the perfusion in step d) is carried out at a pressure of 220 to 240 mmHg, preferably at a pressure of 230 mmHg. 148. The method of any one of the preceding embodiments, wherein the permeabilization solution comprises a cyclodextrin derivative as identified in any one of embodiments 2 to 48. 149. The method of any one of the preceding embodiments, wherein the labeling solution comprises a cyclodextrin derivative of any one of embodiments 2-48. 150. The method of any one of the preceding embodiments, wherein the labeling solution is the composition specified in any one of embodiments 233-242, comprising the fluorescent dye-containing labeling agent.

[0037] 151. The method of any one of the preceding embodiments, wherein the animal tissue is from a mammal. 152. The method of any one of the previous embodiments, wherein the animal tissue is from a non-human mammal or a human. 153. The method of any one of the preceding embodiments, wherein the animal tissue is from a rodent. 154. The method of any one of the preceding embodiments, wherein the animal tissue is from a mouse. 155. The method of any one of the preceding embodiments, wherein the animal tissue is a whole mouse. 156. The method of any one of the preceding embodiments, wherein the animal tissue is pig brain. 157. The method of any one of the preceding embodiments, wherein the animal tissue is a whole organ or a portion thereof. 158. The target molecule labeled with the labeling agent in step c) (i) a structure present in said fixed animal tissue, preferably a protein, lipid, DNA or RNA, more preferably a protein present in said fixed animal tissue; or (ii) a primary antibody bound to a structure present in the fixed animal tissue, preferably a protein, lipid, DNA or RNA, more preferably a protein present in the fixed animal tissue; 3. The method of any one of the preceding embodiments, wherein 159. The method according to any one of the previous embodiments, wherein the animal tissue comprises a cancer and the target molecule labeled by the labeling agent 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. 160. The method according to any one of the preceding embodiments, wherein the animal tissue contains cancer metastasis and the target molecule labeled by the labeling agent 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.

[0038] 161. The method of any one of the preceding embodiments, wherein the animal has been treated with a biopharmaceutical, the animal tissue contains the biopharmaceutical, and the biopharmaceutical is the target molecule that is labeled with the labeling agent in step c), or the biopharmaceutical is labeled with a further fluorescent dye ex vivo, or the biopharmaceutical is itself fluorescent. 162. The method of embodiment 161, wherein the biopharmaceutical is a small molecule. 163. The method of embodiment 161, wherein the biopharmaceutical is a therapeutic protein. 164. The method of embodiment 161, wherein the biopharmaceutical is a therapeutic antibody. 165. The method of any one of the previous embodiments, which is not a method for the treatment of the human or animal body by surgery or therapy, and which is not a diagnostic method performed on the human or animal body. 166. The method according to any one of the previous embodiments, which is an ex vivo method. 167. A method according to any one of the previous embodiments, wherein the volume of the animal tissue for fluorescence microscopy obtained in step d) is smaller than the volume of the fixed animal tissue used in step b). 168. The method according to embodiment 167, wherein the volume of the animal tissue for fluorescence microscopy obtained in step d) is 40% to 75% smaller than the volume of the fixed animal tissue used in step b). 169. Before labeling target molecules in fixed animal tissue with a labeling solution, the following steps are performed: c.1) contacting the fixed animal tissue with a primary antibody capable of binding to a structure present in said fixed animal tissue, preferably a protein, lipid, DNA or RNA, more preferably a protein present in said fixed animal tissue wherein said fluorescent dye-containing labeling agent is capable of binding to said primary antibody. 170. The method of embodiment 169, wherein step c.1) is carried out by perfusing the fixed animal tissue with a solution containing the primary antibody.

[0039] 171. The method according to embodiment 169 or 170, wherein step c.1) is carried out by perfusing the fixed animal tissue with a primary labeling solution comprising a primary antibody and a cyclodextrin derivative as defined in any one of embodiments 2 to 48. 172. The method of any one of embodiments 169 to 171, wherein the primary antibody is not conjugated to a fluorescent dye and is preferably selected from those listed in Tables 2 and 3. 173. The method of any one of embodiments 169-172, wherein the primary antibody is present in a composition as defined in any one of embodiments 233-242. 174. The method of any one of embodiments 169 to 173, wherein the molecular weight of the primary antibody is determined in any one of embodiments 80 to 97. 175. The method of any one of embodiments 169-174, wherein the primary antibody is IgG, IgA, IgM, IgD or IgE. 176. The method of any one of embodiments 169-175, wherein the primary antibody is an IgG. 177. The method of embodiment 176, wherein the primary antibody is IgG1, IgG2, IgG3, or IgG4. 178. The method according to any one of embodiments 169 to 177, wherein the primary antibody is a rabbit or rat antibody. 179. The following: removing heme from the fixed animal tissue, and treating the tissue with a permeabilization and blocking solution to decolorize, permeabilize, and block the tissue; and The method of any one of the preceding embodiments, comprising labeling a target molecule in the fixed animal tissue with a labeling solution comprising a cyclodextrin derivative and a fluorescent dye-containing labeling agent capable of binding to the target molecule, preferably in this order, to obtain fixed animal tissue labeled with the fluorescent dye-containing labeling agent, wherein the molecular weight of the fluorescent dye-containing labeling agent is greater than 100 kDa.

[0040] 180. The following: Performing decolorization, permeabilization, and blocking by removing heme from the fixed animal tissue, treating with a permeabilization and blocking solution; Contacting the fixed animal tissue with a primary labeling solution containing a cyclodextrin derivative and a primary antibody capable of binding to a structure present in the fixed animal tissue, preferably a protein, lipid, DNA, or RNA, more preferably a protein present in the fixed animal tissue; Labeling the primary antibody bound to the structure in the fixed animal tissue with a secondary labeling solution containing a cyclodextrin derivative and a fluorescent dye-containing labeling agent capable of binding to the primary antibody to obtain the fixed animal tissue labeled with the fluorescent dye-containing labeling agent, The method according to any one of the above embodiments, preferably including in this order, wherein the molecular weight of the fluorescent dye-containing labeling agent exceeds 100 kDa. 181. The method according to embodiment 179 or 180, wherein the solution for heme removal, the permeabilization solution, and the blocking solution are the same solution ("pretreatment solution"). 182. The method according to embodiment 181, wherein the composition of the pretreatment solution is the composition of the blocking solution specified in any one of embodiments 49 to 55. 183. The method according to any one of embodiments 169 to 182, wherein the primary labeling solution contains a composition specified in any one of embodiments 233 to 242 and includes a primary antibody. 184. The method according to any one of embodiments 169 to 183, wherein the labeling with the fluorescent dye-containing labeling agent is performed using a secondary labeling solution containing a fluorescent dye-containing labeling agent and a composition specified in any one of embodiments 233 to 242. 185. The method according to any one of embodiments 179 to 184, further including demineralizing the fixed animal tissue using a demineralizing solution before performing decolorization, permeabilization, and blocking. 186. The method according to any one of embodiments 179 to 185, further including subjecting the fixed animal tissue labeled with the fluorescent dye-containing labeling agent to a permeabilization treatment solution using a clarification solution containing an organic solvent after labeling (if any). 187. An animal tissue or animal body obtainable by the method for preparing animal tissue for fluorescence microscopy described in any one of the preceding embodiments, wherein the animal tissue contains the target molecule labeled with the fluorescent dye-containing labeling agent. 188. The animal tissue of embodiment 187, wherein said animal tissue is a whole rodent, preferably a whole mouse. 189. The animal tissue of embodiment 187, wherein the animal tissue is a whole organ or a part thereof. 190. The animal tissue of embodiment 189, wherein said animal tissue is a whole organ of a mammal.

[0041] 191. The animal tissue of any one of embodiments 187-190, having a size of at least 1 mm length x at least 1 mm width x at least 1 mm height; preferably at least 5 mm length x at least 5 mm width x at least 5 mm height, more preferably at least 1 cm length x at least 1 cm width x at least 1 cm height. 192. The animal tissue according to any one of embodiments 187 to 191, wherein the size of the animal tissue is a tissue block of 2x2x2 cm. 193. An animal tissue according to any one of the preceding embodiments, wherein when the target molecules in the animal tissue are labeled with the fluorescent dye-containing labeling agent and analyzed by fluorescence microscopy, preferably light-sheet fluorescence microscopy, all of the target molecules can be detected with single-cell resolution, regardless of their location in the animal tissue. 194. A method for analyzing an animal tissue or body according to any one of embodiments 187 to 193, the method comprising: i) analyzing the tissue by fluorescence microscopy to detect the fluorescence of the fluorescent dye in the animal tissue. 195. Furthermore, the following: ii) visualizing the detected fluorescence of said fluorescent dye to obtain an image, preferably a three-dimensional image, of said animal tissue. 196. The analytical method of embodiment 195, wherein the image has single-cell resolution throughout the animal tissue. 197. The analytical method according to any one of the previous embodiments, wherein the thickness of the animal tissue is 20 cm or less, preferably 10 cm or less, more preferably 5 cm or less. 198. The analytical method according to any one of the preceding embodiments, wherein the thickness of the animal tissue is 2 cm or less, preferably 1.5 to 2 cm. 199. The analytical method according to any one of the preceding embodiments, further comprising, prior to step i), a method according to any one of embodiments 1 to 186. 200. The method of analysis according to any one of the previous embodiments, wherein the fluorescence microscopy is selected from the group consisting of light sheet fluorescence microscopy, epifluorescence microscopy, multiphoton microscopy and confocal fluorescence microscopy.

[0042] 201. The method of analysis according to any one of the previous embodiments, wherein the fluorescence microscopy is fluorescence microscopy, preferably light sheet fluorescence microscopy. 202. The analysis method of any one of the preceding embodiments, further comprising, after step i), iii) dissecting the tissue region of interest, iv) rehydrating the dissected tissue region of interest, and v) further analyzing the dissected tissue region of interest. 203. An analytical method according to embodiment 202, wherein in step v), the dissected tissue area of ​​interest is further analyzed by antibody-based immunostaining, or by gene profiling, preferably gene profiling by RNAseq, or by proteomics, preferably proteomics by mass spectrometry. 204. The method of analysis according to embodiment 202 or 203, wherein the dissected tissue area of ​​interest contains metastases, preferably metastases with a size of less than 200 tumor cells, more preferably less than 100 metastases, even more preferably less than 75 metastases, even more preferably less than 50 metastases, even more preferably less than 25 metastases. 205. A method for detecting metastasis, comprising the method of analyzing animal tissue according to any one of the previous embodiments. 206. The method of embodiment 205, which is a method for detecting metastasis in an animal tissue at single-cell resolution throughout the animal tissue. 207. A method for detecting metastasis according to any one of the preceding embodiments, wherein the animal tissue contains cancer metastasis and the target molecule labeled by the labeling agent is a structure, preferably a protein, present in the cancer. 208. A method for analyzing the biodistribution of a biopharmaceutical, comprising analyzing an animal tissue according to any one of the preceding embodiments, wherein the animal has been treated with a biopharmaceutical identified in embodiment 161, the animal tissue contains the biopharmaceutical, and the biopharmaceutical is the target molecule labeled with the labeling agent. 209. The method of embodiment 208, wherein the biopharmaceutical is a therapeutic protein, preferably a therapeutic antibody. 210. The method of embodiment 208, wherein the biopharmaceutical is a nanoparticle.

[0043] 211. A method for analyzing the biodistribution of nanoparticles, comprising the method of analyzing animal tissue according to any one of the preceding embodiments, wherein the animal has been treated with nanoparticles and the animal tissue contains the nanoparticles, wherein the nanoparticles are the target molecules labeled with the labeling agent and / or are selected from fluorescent dye-conjugated nanoparticles or nanoparticles that are themselves fluorescent. 212. The method for analyzing the biodistribution of nanoparticles of embodiment 211, wherein the nanoparticles carry a drug or are a drug. 213. A method for testing neurodegeneration, comprising a method for analyzing animal tissue according to any one of the preceding embodiments, wherein the animal tissue contains neurons. 214. A method for testing neurodegeneration according to embodiment 213, wherein neurons in the animal tissue are fluorescently labeled, preferably by expression of a fluorescent protein. 215. A method for testing neurodegeneration according to any one of the previous embodiments, wherein testing for neurodegeneration comprises analysis of nerve axon blebbing. 216. A method for testing neuroinflammation, comprising a method for analyzing animal tissue according to any one of the preceding embodiments, wherein the animal tissue contains neurons. 217. A method for testing neuroinflammation according to embodiment 216, wherein the immune cells in the animal tissue are fluorescently labeled, preferably by expression of a fluorescent protein. 218. A method for testing neuroinflammation according to any one of the preceding embodiments, comprising testing immune cell activation by analyzing the signal intensity and / or cell number of fluorescently labeled immune cells. 219. A method for examining meningeal lymphatic vessels, comprising a method for analyzing animal tissue according to any one of the preceding embodiments, wherein the animal tissue preferably comprises an intact mouse head. 220. A method for examining meningeal lymphatic vessels according to embodiment 219, wherein the meningeal lymphatic vessels are preferably fluorescently labeled with a marker protein or tracer, such as ovalbumin.

[0044] 221. A method for testing meningeal lymphatic vessels according to any one of the previous embodiments, wherein the meningeal lymphatic vessels contain the target molecule. 222. Use of a cyclodextrin derivative to improve labeling of said target molecule in fixed animal tissue or in a whole animal with a fluorescent dye-containing labeling agent. 223. The use of embodiment 222, wherein the fluorescent dye-containing labeling agent is a fluorescent antibody. 224. The use according to embodiment 222 or 223, wherein the molecular weight of the fluorescent dye-containing labeling agent is greater than 100 kDa. 225. The use according to any one of embodiments 222 to 224, wherein the fluorescent dye-containing labeling agent is as specified in any one of embodiments 80 to 115. 226. The use according to any one of embodiments 222-225, wherein the cyclodextrin derivative is as defined in any one of embodiments 2-48. 227. The use according to any one of embodiments 222-226, wherein the cyclodextrin derivative is not methyl-β-cyclodextrin with a degree of substitution of 1.8. 228. Use according to any one of embodiments 222-227, wherein the cyclodextrin derivative is not a methyl-β-cyclodextrin with a degree of substitution <2.0. 229.R 2 , R 3 and R 6 The use according to any one of embodiments 226-228, provided that DS≧2 when each is selected from H and CH3. 230.R 2 and R 6 is CH3 and R 3 is H; or R 2 , R 3 and R 6 are each independently selected from H and CH2CH(OH)CH3, and / or DS≧0.9.

[0045] 231. 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, heptakis(2,6-di-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, γ-cyclodextrin and α-cyclodextrin; more preferably (2-hydroxypropyl)-β-cyclodextrin or heptakis(2,6-di-O-methyl)-β-cyclodextrin; most preferably heptakis(2,6-di-O-methyl)-β-cyclodextrin. 232. The use according to any of embodiments 222 to 231, which is not a method for the treatment of the human or animal body by surgery or therapy, and which is not a diagnostic method performed on the human or animal body. 233. A composition comprising an antibody, preferably having a molecular weight of more than 100 kDa, and a cyclodextrin derivative. 234. The composition according to embodiment 233, wherein the cyclodextrin derivative is as defined in any one of embodiments 2 to 48. 235.R 2 , R 3 and R 6 is each selected from H and CH3, then DS≧2. 236.R 2 and R 6 is CH3 and R 3 is H; or R 2 , R 3 and R 6 are each independently selected from H and CH2CH(OH)CH3, and / or DS > 0.9. 237. The composition according to any one of embodiments 233-236, wherein the antibody is a fluorescent dye-containing labeling agent, preferably as specified in any one of embodiments 80-115. 238. The composition according to any one of embodiments 233 to 236, wherein the antibody is preferably a primary antibody as defined in any one of embodiments 172 to 178. 239. In an aqueous buffer, preferably phosphate buffered saline (PBS), further comprising: animal serum, preferably mammalian serum, more preferably goat serum; a zwitterionic detergent, preferably CHAPS or CHAPSO, more preferably CHAPS; a non-ionic surfactant, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100; an organic solvent, preferably a water-miscible solvent, more preferably DMSO; amino acids, preferably glycine; 239. The composition of any one of embodiments 233 to 238, comprising one or more, preferably all, of: 240. When the component is present in an aqueous buffer, the following concentrations are present: Cyclodextrin derivative: 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v; Animal serum: 0.5-12, preferably 0.75-10, more preferably 1-3% 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; The buffer concentration of the aqueous buffer solution is 0.05 to 0.2 M, preferably 0.08 to 1.2 M, more preferably 0.M; 240. The composition of embodiment 239, wherein

[0046] 241. The following: 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v of a cyclodextrin derivative 0.5-12, preferably 0.75-10, more preferably 1-3% v / v goat serum; 5-15, preferably 7.5-12.5, more preferably 10% w / v CHAPS; 0.5 to 4, preferably 1 to 3, more preferably 2% w / v Triton X-100; 5 to 20% w / v, preferably 7.5 to 15% w / v, more preferably 10% w / v DMSO; and 0.5 to 2, preferably 0.75 to 1.5, more preferably 1% w / v glycine; 0.05 to 0.2 M, preferably 0.08 to 1.2 M, more preferably 0.1 M phosphate buffered saline; 241. The composition of any one of embodiments 233 to 240, comprising: 242. The composition according to any one of embodiments 233 to 241, having a pH value of 7.7 to 7.4, preferably 7.2. Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Brief explanation of the drawings]

[0047] [Figure 1] Development of wild-type DISCO and single-antigen total staining in mice. (a) Structures of cyclodextrins (CDs) with different substituents: 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), and CD6 (succinyl-β-cyclodextrin). (b) Measurement of supernatant cholesterol concentration after 7 days of incubation of 25 mg mouse liver sections with control, CD1, CD2, CD4, CD5, CD6, γ-cyclodextrin (CD7), and α-cyclodextrin (CD8) in different CD-containing buffers. (c) Methylene blue staining of a single hemisphere of mouse brain after permeabilization with different CD-containing solutions. It is shown that CD5 significantly enhanced tissue permeabilization for dye transfer compared to other antibodies. (d) Dynamic light scattering (DLS) of the size distribution of TH antibodies in CD5-containing and CD5-free solutions. (e) Dark color coding indicates the projection of pan-neuronal marker PGP-9.5+ neurons at different z-levels in a 2.0 cm-thick mouse whole body. (f, g) Details of innervation throughout hard (f, vertebrae) and soft (g, adipose) tissues. (h) Manually segmented vagus nerves (green) innervating the liver (cyan), spleen (magenta), intestine (red), and kidney (yellow) are highlighted with specific pseudocolors. (i) Whole mouse stained with lymphatic marker LYVE1 (yellow). (j) Lymphatic element (LYVE1) staining was detected in the brain parenchyma of a mouse. (k) Mouse brain stained with two different lymphatic markers (LYVE1 and podoplanin) to identify lymphatic endothelial cells found in different brain regions. [Figure 2]Figure 1: Examination of the spatial relationships of different physiological systems using wildDISCO. (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), illustrating the context of neuro-immune interactions in the visceral organs. (b) Branches of the sympathetic nervous system (TH, green) connect different regions of the intestine. CD45+ cells (magenta) accumulate along segments of the vagus nerve, particularly in the inferior mesenteric plexus. (c) Higher magnification of the labeled area in (a), showing the colocalization of sympathetic nerve fibers and immune cells on the intestinal wall. (d) Maximum intensity projection of a whole mouse stained with TH (green) and LYVE1 (yellow). (e-g) Representative 2D optical sections of hindlimb LNs stained with TH, PGP9.5, CD45, Prox1, and LYVE1, as shown in these images, demonstrating that LNs are innervated by peripheral nerves with immunoregulatory capabilities. (h, i) Representative 3D representation of the myenteric nerve lattice network in WT and germ-free mice by immunostaining with an antibody against PGP9.5. In germ-free mice, the myenteric nerve lattice network appears less ganglionic and disorganized. (l) The density of the PGP9.5 myenteric plexus was quantified. n = 5; mean ± SD; **p < 0.01 (Student's t-test). [Figure 3] Figure 3: Overview of wildDISCO immunostaining buffer and quantification of permeabilization efficacy. (a) Diagram of the major chemical components involved in the wildDISCO immunostaining buffer. (b) Profile plots along each mouse brain dimension from Figure 1c. (c) Methylene blue staining of individual hemispheres of mouse brain after permeabilization with different CD-containing solutions and imaging of different brain cryosections. CD5 has been shown to strongly enhance tissue permeabilization compared to the others. n=4. (d) Profile plots along each cryosection of the mouse brain from Figure 3c. [Figure 4]Figure 1 shows wildDISCO immunostaining of PGP9.5 in whole mouse body. (a) Maximum projection of the peripheral nervous system from a 4-week-old mouse stained with PGP9.5 antibody using light-sheet microscopy. (b) Examples of positive PGP9.5 staining in various organs (heart, spleen, liver, and intestine) with higher magnification. (c) Visualization of peripheral nerve innervation on multiple organs (adrenal gland (green), kidney (magenta), and ureter (cyan)). [Figure 5] Figures showing wildDISCO immunostaining of LYVE1 in the whole mouse body. (h)-(i) 3D reconstruction of the intestinal lymphatic network using Syglass reconstruction software. [Figure 6] Figure 1 shows nerve-immune cell interactions in the intestine. (a) 3D reconstruction of Peyer's patches in the intestine stained with CD45 (green) and innervated by TH+ sympathetic nerves (magenta) visualized with Syglass software. (b) The sympathetic nerve marker TH (green) and the immune cell marker CD45 (magenta) on the intestinal wall were shown with Imaris software. [Figure 7] Nerve-lymphatic vessel interactions in the intestine: (a) PGP9.5 nerve fibers (magenta) interacting with Prox1 lymphatic vessels (green). (b) TH sympathetic nerves (green) and LYVE1 lymphatic vessels (magenta). [Figure 8] Nerve-lymphatic interactions in the kidney. (a) TH sympathetic neurons (green) innervated LYVE1 lymphatic vessels (magenta) in the kidney. (b) PGP9.5 pan-neuronal marker (green) combined with Prox1 lymphatic vessel marker (magenta). [Figure 9] Figure 1 shows the influence of the microbiota on the sympathetic nervous system in mice. (ab) Myenteric nerve lattice network of WT and germ-free mice immunostained with an antibody against TH. Higher magnification of the areas marked by the white and yellow boxes, respectively. [Figure 10]WildDISCO was applied to a cancer metastasis model. Tyrosine hydroxylase (TH) co-stained with breast cancer cells MDA-MB-231. Cancer cells were clearly visible in close proximity to TH+ nerves. [Figure 11] WildDISCO clearly demonstrates the entire arterial network in mice. Alpha-smooth muscle actin (alpha-SMA) was applied to examine the distribution of arteries on a whole-body scale. Larger-diameter arteries branched into arterioles penetrating from the meninges to the corpus callosum (A). The distribution of arterioles was symmetric and regular in the eyes, olfactory bulbs, meninges, and cerebrum (B). αSMA, a defining characteristic of mature cardiac fibroblasts known as myofibroblasts, clearly visualized coronary arteries and their associated branches in the heart (C). αSMA was detected in the liver sinusoidal spaces and portal and central veins (D). The splenic artery branched into arterioles forming a reticular capillary network (E). Spike-like vascular structures were also observed in the kidney and lung (F and G). Transverse sections of dorsal α-SMA-labeled mice revealed the distribution of arteries in several organs, particularly arterioles in the spinal cord (H). [Figure 12] FIG. 1 shows a schematic diagram of the wildDISCO pipeline (an exemplary series of steps (1) to (6)); preferred conditions for each step). [Figure 13] Comparison of different clearing methods for whole-body mouse antibody staining. (a) Optical 2D light-sheet microscopy images of a mouse whole body stained with synapsin 1 using wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods. Scale bar: 5000 μm. (b) Representative 2D optical images of the mouse hind limb and spinal cord using wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods. Scale bar: 300 μm. n=3. (c) Quantification of antibody penetration depth into the mouse hind limb and spinal cord using wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods. [Figure 14]Comparison of different clearing methods using synapsin-1 staining. (a-c) Representative 2D optical images of forelimbs, livers, and kidneys stained with synapsin-1 antibody by wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS methods, respectively. n=3. (df) Quantification of the penetration depth of synapsin-1 antibody into forelimbs (d), livers (e), and kidneys (f) of mice treated with wildDISCO, vDISCO, iDISCO, uDISCO, and PEGASOS, respectively. [Figure 15] Visualization and analysis of tumor-associated TLS in a tumor metastasis model using wildDISCO and deep learning. (a) 3D rendering of a mouse bearing 4T1 cell metastasis using light-sheet microscopy imaging in the ventral field. TLS are detected and masked in magenta, tumor cells are masked in yellow, and the background is cyan. Higher magnifications show TLS in detail. Scale bar, 2000 μm. (b-1) Exemplary images of TLS in a tumor-bearing mouse stained with CD23 in red (b, c), CD3 in green, and CD23 in magenta (d-g). TLS were masked in magenta in the primary tumor (h), intestine (i, j), and lung (k, l). Scale bars: 500 μm (d, h, k); 150 μm (e, f, g, j); 400 μm (i), and 200 μm (l). Quantification of the spatial correlation between TLS and metastasis across the mouse. (m) Quantification of metastatic volume throughout the mouse. (n) Quantification of metastatic density in the lung and intestine. n=4 mice. (o) Quantification of TLS distribution throughout the mouse. n=4 mice. (p) Quantification of TLS volume throughout the mouse. (q,r) Quantification of the distance to the nearest neighboring TLS (q) and the distance between the metastasis and the nearest TLS (r). (s) Metastatic volume to the nearest TLS. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention will be described in detail below. Definitions and General Techniques Unless otherwise defined below, terms used in the present invention shall be understood by their ordinary meanings known to those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. As used herein, such publications, patents, and patent applications are preferably identified by the name of the first author and the year or number of publication. For each reference identified in this manner, each corresponding reference, including the specific source of the publication (e.g., the name and volume of the scientific journal, etc.), can be found in "References." The materials, methods, and examples are illustrative only and, unless otherwise specified, are not intended to be limiting.

[0049] The term "fluorescent dye" as used herein is not particularly limited. For example, the fluorescent dye may be a synthetic compound such as a fluorescent protein or a synthetic organic compound. Preferably, the fluorescent dye used in accordance with the present invention can emit fluorescence in the red or infrared range, more preferably in the far-red or near-infrared range. Preferred wavelengths of emission maxima of the fluorescent dyes used in accordance with the present invention are as set forth in the preferred embodiments of the present invention. Non-limiting examples of fluorescent dyes capable of fluorescing in the far-infrared or near-infrared range and that can be used in accordance with the present invention are known in the art and are described, for example, in Hong et al. (2017), "Near-infrared fluorophores for biomedical imaging." Nature Biomedical Engineering 1, 0010, which is incorporated by reference in its entirety for all purposes. Fluorescent dyes that can fluoresce in the far-red or near-infrared region and that can be used in accordance with the present invention are commercially available and preferably include, for example, ATTO dyes such as ATTORhol3, ATTO594, ATTO550, ATTO610, ATTO620, ATTORhol4, ATTO633, ATTO647, ATTO647N, ATTO655, ATTOOxal2, ATTO665, ATTO680, ATTO700, ATTO725, and ATTO740, and Alexa Fluors 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® 790. These include Fluor® dyes 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 may be used in the methods of the invention.

[0050] Labeling with fluorescent dye-containing labeling agents having a molecular weight of 100 kDa or less is also contemplated. That is, although the present invention is particularly advantageous for improving labeling of target molecules in fixed animal tissues or whole animals with fluorescent labeling agents (such as antibodies) having a molecular weight of more than 100 kDa, it is also contemplated that equivalent improvements can be achieved using the cyclodextrin derivatives described herein to improve labeling with certain agents having a molecular weight of 100 kDa or less (e.g., antibody fragments conjugated to fluorescent dyes or other dyes).

[0051] The animal tissues that can be used in the uses and methods of the present invention are not particularly limited. They can be derived from any animal species. In a preferred embodiment of the present invention, the animal tissue can be derived from a non-human mammal or a human. Preferably, the animal tissue derived from a non-human mammal is derived from a rodent, more preferably from a mouse. Even 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 a part thereof, preferably a human organ or a part thereof. The animal tissue may contain a recombinantly expressed fluorescent protein (e.g., GFP, YFP, and mCherry) that can be used as a target molecule. For example, the animal from which the animal tissue is obtained can be an animal (e.g., a mouse) transplanted with cancer cells expressing the recombinant fluorescent protein. The size of the fixed animal tissue used in the uses and methods of the present invention is preferably at least 1 cm long x at least 1 cm wide x at least 1 cm high.

[0052] The decolorizing 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 with the decolorizing step and does not include fixation of the animal tissue. Therefore, in a preferred embodiment, all methods and uses of the present invention may preferably not be methods for treating the human or animal body by surgery or therapy, and may not be diagnostic methods performed on the human or animal body. In a related preferred embodiment according to all other embodiments of the present invention, the methods or uses of the present invention are ex vivo methods and uses. Therefore, the methods of the present invention may preferably be performed outside of living animals.

[0053] Fixed animal tissue suitable for the methods of the present invention can be easily identified by those skilled in the art. For example, for PFA 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; i.p.) and then intracardially perfused with heparinized 0.1 M PBS (10 U / mL heparin, Ratiopharm; 100-125 mmHg pressure using a Leica Perfusion One system) at room temperature for 5-10 minutes until blood is flushed out. After this procedure, tissue can be fixed for 10-20 minutes using, for example, 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) (Morphisto, 11762.01000). If vasculature staining is desired, animal tissues, such as mice (e.g., whole mice), can be intracardially perfused with 20 ml of PBS (heparin-free) containing 0.5 mg of FITC-conjugated lectin (EY Laboratories, F-2101-5) before proceeding with 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; i.p.) and then intracardially perfused with heparinized 0.1 M PBS (10 U / ml heparin, Ratiopharm; 100–125 mmHg pressure using a Leica Perfusion One system) at room temperature for 5–10 minutes until the blood is flushed out. This procedure can be followed by fixation with the injection of 40–50 ml of PaXgene fixative solution. If preservation of PaXgene-fixed animals is required before further processing, the tissue can be kept in PaXgene stabilization solution. If vasculature staining is desired, animal tissues such as mice (e.g., whole mice) can be intracardially perfused with 20 ml of PBS (heparin-free) containing 0.5 mg of FITC-conjugated lectin (EY Laboratories, F-2101-5) before proceeding with PFA fixation. The skin can then be carefully removed, or left intact if the animal is hairless, and the body can be post-fixed in 4% PFA at 4°C for 1 day and transferred to 0.1 M PBS.The method of the present invention can be initiated immediately or whole mice can be stored, preferably at 4°C in PBS for up to 4 weeks or in PBS containing 0.05% sodium azide (Sigma, 71290) for up to 6 months.

[0054] As used herein, the term "animal tissue for fluorescence microscopy" and the like is meant to indicate that the respective animal tissue is suitable for fluorescence microscopy. Similarly, the term "for heme removal" in reference to a solution refers to any solution suitable for heme removal. According to the present invention, heme removal is not limited to a specific mechanism, as long as it removes heme from tissue and / or decolorizes heme. For example, an amino alcohol suitable for heme removal, such as N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine, can be used, for example, as shown in a preferred embodiment. The amino alcohol competes with hemoglobin for heme binding and can be used to remove heme from hemoglobin in and from tissue. Alternatively, benzyl peroxide can be used.

[0055] The term "fluorochrome-containing labeling agent" as used herein is not particularly limited, as long as it is suitable for labeling a target molecule in bleached, fixed animal tissue, can bind to the target molecule, and has a molecular weight of more than 100 kDa (e.g., 110 kDa or more, 120 kDa or more, 130 kDa or more, or 140 kDa or more). In a preferred embodiment, the fluorescent dye-containing labeling agent is an antibody conjugated to the fluorescent dye.

[0056] As used herein, the term "target molecule" refers to any target molecule in a tissue. It is understood that an appropriate target molecule can be selected for a given application of the method of the present invention, such as a biomedical application. The target molecule can be, for example, an endogenous molecule of the animal (e.g., a marker protein for a disease such as cancer) or a recombinant molecule such as a recombinant protein. For example, in a preferred embodiment according to the present invention, when the animal from which the animal tissue is obtained is an animal (e.g., a mouse) transplanted with cancer cells expressing the recombinant fluorescent protein, the fluorescent protein can be the target molecule. Alternatively, the target molecule can be any other structure (e.g., an exogenous molecule) present in the fixed animal tissue, such as a protein, lipid, DNA, or RNA present in the fixed animal tissue. More preferably, the target molecule can be a protein present in the fixed animal tissue, such as a therapeutic antibody or a primary antibody bound to a tissue antigen in the fixed animal tissue. The primary antibody that can be used in the present invention is not particularly limited. In one embodiment, the primary antibody is selected from the group consisting of an anti-tyrosine hydroxylase antibody, an anti-PGP9.5 antibody, an anti-S100 beta antibody, an anti-neurofilament M antibody, an anti-alpha smooth muscle actin antibody, an anti-collagen IV antibody, an anti-Prox1 antibody, an anti-LYVE1 antibody, an anti-Iba1 antibody, and an anti-CD45 antibody. Preferably, the primary antibody is not a rabbit anti-neurofilament NF-M antibody.

[0057] The term "labeling a target molecule" as used herein should also be understood to mean that more than one target molecule may be labeled by the method of the present invention. Thus, in a preferred embodiment according to the present invention, more than one target molecule, for example, two or three target molecules, are labeled. For example, in a preferred embodiment according to the present invention, when the animal from which the animal tissue was obtained is an animal (e.g., a mouse) transplanted with cancer cells expressing the recombinant fluorescent protein, the fluorescent protein may be the first target molecule, and a biopharmaceutical against cancer (e.g., a therapeutic antibody against cancer) administered to the animal may be the second target molecule. Thus, in a preferred embodiment such as this embodiment, the method for detecting metastasis according to the present invention and the method for analyzing the biodistribution of a biopharmaceutical can be performed together.

[0058] As used herein, the term "perfusion at a pressure" refers to a pressure measurable at the tissue entry point. Pressure can be measured by any method known in the art. Preferably, it is measured with a pressure gauge, more preferably a Kkmoon Digital Manometer Pressure Gauge Manometer (HT-1891). When using a Kkmoon Digital Manometer Pressure Gauge Manometer (HT-1891), a two-head connector (B. Braun Dicofix® C Dreiwegehahn, 16494C) can be inserted into the pumping channel and connected to the manometer. The pumping channel can be set using a transcardial perfusion needle (Leica, 39471024), and pressure can be measured at the pumping rate used in this method (if the reading is stable).

[0059] As used herein, the term "permeabilization solution" refers to a solution suitable for permeabilizing animal tissue. Such solutions are well known in the art and can be easily selected by those skilled in the art, for example, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; CAS number: 75621-03-3), CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate; CAS number: 82473-24-3), IGEPAL CA-630 (octylphenoxypoly(ethyleneoxy)ethanol; CAS number: 68412-54-4; linear formula: (CHO)C 14 H 22 Suitable surfactants for permeabilization include (1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (CAS number: 9002-93-1; linear formula: t-Oct-C6H4-(OCH2CH2)xOH (e.g., average x = 9.5)) and / or Triton X-100 (4 M).

[0060] As used herein, the terms "labeling solution," "immunostaining solution," or "immunostaining buffer" refer to a solution containing a fluorescent dye-containing labeling agent, unless otherwise specified. Typically, a labeling solution contains one or more surfactants, an organic solvent, and a labeling agent. Advantageously, the labeling solution comprises one or more, preferably all, of the following components in an aqueous buffer, preferably phosphate buffered saline (PBS): animal serum, preferably mammalian serum, more preferably goat serum; a zwitterionic detergent, preferably CHAPS or CHAPSO, more preferably CHAPS; a non-ionic detergent, preferably Triton X-100 or IGEPAL CA-630, more preferably Triton X-100; an organic solvent, preferably a water-miscible solvent, more preferably DMSO; and / or an amino acid, preferably glycine. Preferably, the permeabilization solution and / or labeling solution used in the present invention further comprises an agent suitable for extracting cholesterol from biological membranes. Without wishing to be bound by any theory, it is hypothesized that insufficient cholesterol extraction from cell membranes may have been a limiting factor in permeabilization in prior art protocols (Mahammad, S. & Parmryd, I. in Methods in Membrane Lipids. (ed. DM Owen) 91-102 (Springer New York, New York, NY; 2015)). Preferred cyclodextrin derivatives are summarized in Table 1 below.

[0061] Table 1. Cyclodextrin derivatives

[0062] [Table 1] TIFF2025536457000004.tif28170 As used herein, the term "total degree of substitution" or "TDS" refers to the average total number of moles of non-hydrogen substituents R2, R3, and R6 per mole of substituted cyclodextrin. TDS can be determined by methods known in the art, such as 1H nmR, ESI-MS, or MALDI-TOF-MS. For a particular substituted cyclodextrin where the non-hydrogen substituents are represented by R, the TDS can be calculated based on the number average molecular weight Mn according to the following formula:

[0063] TDS = [Mn-M(unsubstituted cyclodextrin)] / [M(R)-1] As used herein, the term "degree of substitution" or "DS" refers to the average number of moles of non-hydrogen substituents R2, R3, and R6 per mole of glucopyranose unit of a cyclodextrin. DS can be determined by methods known in the art. For a substituted cyclodextrin, the DS can be calculated based on the total degree of substitution (TDS) as defined above according to the following formula:

[0064] DS=TDS / m 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).

[0065] A preferred cyclodextrin derivative is one in which m=7, i.e., β-cyclodextrin. The cyclodextrin derivative is preferably selected from (2-hydroxypropyl)-β-cyclodextrin, triacetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin, succinyl-β-cyclodextrin, γ-cyclodextrin, and α-cyclodextrin; more preferably, (2-hydroxypropyl)-β-cyclodextrin or heptakis(2,6-di-O-methyl)-β-cyclodextrin; most preferably, heptakis(2,6-di-O-methyl)-β-cyclodextrin. The cyclodextrin derivative is preferably not a methyl-β-cyclodextrin, for example, it is not a methyl-β-cyclodextrin having a degree of substitution of <2.0, for example, 1.8. Particularly preferred cyclodextrin derivatives are those in which R2, R6 = CH3 and R3 = H, more preferably heptakis(2,6-di-O-methyl)-β-cyclodextrin (i.e., CD5); or R 2 , R 3 and R 6 is selected from H and CH2CH(OH)CH3, more preferably (2-hydroxypropyl)-β-cyclodextrin (e.g., CD2); most preferably heptakis(2,6-di-O-methyl)-β-cyclodextrin.

[0066] Preferably, the cyclodextrin derivative used in the present invention is capable of extracting cholesterol from mouse liver tissue such that the cholesterol concentration after 7 days of incubation is ≧14, preferably ≧30, more preferably ≧50, and most preferably ≧70 μM, as measured under the conditions described in the Examples below, "Screening of cyclodextrin-containing buffers for cholesterol extraction."

[0067] Preferably, the permeabilization solution used in the present invention further comprises an agent for loosening the collagen network, such as trans-1-acetyl-4-hydroxy-L-proline. When a permeabilization solution is used in a particular step of the method of the present invention, it should be understood that this does not exclude that other solutions, such as solutions used in previous steps of the method, may also contribute to and improve permeabilization. For example, a solution for removing heme may be a solution that contributes to permeabilization.

[0068] The solutions or compositions described herein, particularly labeling solutions, typically comprise a zwitterionic detergent such as CHAPS or CHAPSO, or a combination of a zwitterionic detergent with a non-ionic detergent such as Triton X-100 or IGEPAL CA-630. Each of these detergent types is believed to enhance permeabilization, and the combination is believed to provide a synergistically enhanced effect, for example, for the following reasons: Detergent properties: Zwitterionic detergents such as CHAPS have positive and negative charges, making them milder and less denaturing to proteins. Nonionic detergents such as Triton X-100 are similarly mild, but have different affinities for various proteins and lipids. Therefore, this combination can solubilize a wider range of biomolecules; Micelle formation: Surfactants work by forming micelles around the hydrophobic (water-insoluble) parts of the molecule, making them soluble in aqueous solutions. The size and properties of the micelles formed by different surfactants can vary, and when used together can more effectively solubilize different types of molecules; Protein solubilization: Different proteins have different conformations and hydrophobic regions. The combined use of these two detergent types is believed to be more effective in solubilizing various proteins without denaturing them, as the detergents can complement each other in stabilizing different proteins.

[0069] Measuring tissue volume according to the present invention can be done by any suitable method known in the art. Preferably, the volume is measured, for example, by measuring the volumetric displacement of fluid by the tissue in a suitable cylinder. Preferably, the method of the present invention further includes a blocking step. The blocking step is carried out by treating the fixed animal tissue with a blocking solution before the labeling step. As used herein, the term "blocking solution" refers to a solution suitable for blocking nonspecific antigen binding of antibodies and may contain, 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 at a concentration of, for example, 1 to 15% v / v, preferably 3 to 10% v / v, more preferably 3% v / v. The blocking solution may further 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 at 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 an aqueous buffer solution, preferably phosphate buffered saline (PBS), with a buffer concentration of 0.05-0.2M, preferably 0.08-1.2M, more preferably 0.1M.

[0070] In a preferred embodiment, treatment with the permeabilization solution is carried out simultaneously with treatment with the blocking solution, for example by simultaneously providing a permeabilization solution (preferably comprising animal serum and a detergent as described above) that is also a blocking solution.

[0071] A particularly preferred method according to the present invention is the following: removing heme from fixed animal tissue, decolorizing, permeabilizing and blocking by treating with a permeabilizing and blocking solution; and The method comprises labeling a target molecule in the fixed animal tissue with a labeling solution comprising a cyclodextrin derivative and a fluorescent dye-containing labeling agent capable of binding to the target molecule, preferably in this order, to obtain fixed animal tissue labeled with the fluorescent dye-containing labeling agent, wherein the molecular weight of the fluorescent dye-containing labeling agent exceeds 100 kDa.

[0072] An even more preferred method according to the present invention comprises the steps of: Removal of heme from fixed animal tissues, decolorization, permeabilization and blocking by treatment with permeabilization and blocking solutions; contacting the fixed animal tissue with a primary labeling solution comprising a cyclodextrin derivative and a primary antibody capable of binding to a structure present in the fixed animal tissue, preferably a protein, lipid, DNA or RNA, more preferably a protein present in the fixed animal tissue; labeling the primary antibody bound to the structure in the fixed animal tissue with a secondary labeling solution containing the cyclodextrin derivative and a fluorescent dye-containing labeling agent capable of binding to the primary antibody and preferably having a molecular weight of more than 100 kDa, thereby obtaining the fixed animal tissue labeled with the fluorescent dye-containing labeling agent; preferably in that order. The solution for removal of heme, the permeabilization solution and the blocking solution are preferably the same solution (the "pretreatment solution"), preferably a blocking solution whose composition is as described hereinabove.

[0073] The primary and secondary labeling solutions preferably have the composition of the labeling solution described above. For example, the primary labeling solution may have the composition of the labeling solution described above in which the antibody is a primary antibody (e.g., an antibody capable of binding to a structure present in fixed animal tissue), and the secondary labeling solution may have the composition of the labeling solution described above in which the antibody is a fluorescent dye-containing labeling agent (e.g., an antibody conjugated to a fluorescent dye) capable of binding to the primary antibody. The pH value of the composition suitable as the labeling solution described herein is preferably 7 to 7.4, more preferably 7.2.

[0074] The term "clearing solution" as used herein refers to a solution suitable for removing animal tissue. The solution is not particularly limited as long as it contains an organic solvent. It is understood that the organic solvent can be easily selected by those skilled in the art based on, for example, its electromagnetic absorption / emission spectrum (particularly, the lack of fluorescence in the visible, red, and near-infrared ranges) so that it is compatible with the method of the present invention. Preferably, the refractive index of the clearing solution containing an organic solvent is similar to that of the animal tissue (e.g., bone), as reflected in preferred embodiments. The clearing solution is particularly advantageous for tissue clearing. Examples of preferred organic solvents that can be used in the clearing solution of the present invention include solvents containing benzyl alcohol, benzyl benzoate, and diphenyl ether, solvents containing ethyl cinnamate, and solvents containing allyl cinnamate.

[0075] Methods for measuring refractive index are well known in the art. Refractive index values ​​referred to herein are values ​​measured at room temperature (i.e., 25°C) and standard atmospheric pressure (i.e., 760 mmHg).

[0076] The term "additional organic solvent" used in the present invention is not particularly limited. It is understood that the solvent will be selected by those skilled in the art to be suitable for dehydration. Examples of such solvents are THF, dichloromethane, and 1,4-dioxane. For example, according to the present invention, a preferred perfusion using an increasing gradient of 0 to 100% by volume of an additional organic solvent can be perfusion using a gradient of 0 to 100% by volume of THF, followed by incubation with dichloromethane. Alternatively, in all embodiments according to the present invention, dichloromethane can be replaced with 1,4-dioxane.

[0077] Further improvements in all other embodiments of the present invention can be achieved by adding a demineralization step, which further improves bone removal. Decalcification chemicals are known, such as solutions containing EDTA, preferably further containing NaHCO3. Preferably, the pH value of the demineralization solution is 8-9. The demineralization step is performed before the decolorization step, or before the labeling step if no decolorization step is performed.

[0078] The term "present in said fixed animal tissue" as used herein in reference to a structure refers to a structure present in the fixed animal tissue. This term does not mean that the structure must be present inside the cells, but rather that the structure may be present on or outside the cells of the fixed animal tissue, for example, on the surface of the cells of the fixed animal tissue or in the extracellular matrix of the fixed animal tissue. As used herein, the term "therapeutic antibody" refers to any therapeutic antibody and therapeutic antibody fragment known in the art. Furthermore, the term is not limited to therapeutic antibodies and therapeutic antibody fragments per se, but also includes conjugates such as antibody-drug conjugates. The term "small molecule" as used herein has its art-recognized meaning. Typically, small molecules used in accordance with the present invention have a molecular weight of <900 Daltons. When the term "comprising" is used in the present invention, it may optionally be replaced with the term "consisting of." The invention is illustrated by the following non-limiting examples.

[0079] [Example] Unless otherwise stated, the following methods were used in the examples. Animals involved in the study For the wildDISCO study, we used the following: 4-week-old mixed-sex wild-type mice (C57BL / 6J, CD1, and Balb / c) purchased from Charles River Laboratories. The animals were housed under a 12 / 12-hour light / dark cycle and provided food and water randomly. The temperature was maintained at 18-23°C and the humidity at 40-60%. Age- and sex-matched germ-free C57BL / 6J mice were purchased from the Technical University of Munich (Institute of Nutrition and Health, Core Facility Gnotobiology) and housed in a germ-free isolation room. After confirming the absence of bacteria in germ-free mice by microbial culture, the mice were used for further experiments. Each antibody was successfully replicated in at least five mice and by at least three different individuals. Animal experiments were performed in accordance with the institutional guidelines of the Ludwig Maximilan University of Munich and the Helmholtz Munich Center German Mouse Clinic after approval by the Ethical Review Board of the Government of Upper Bavaria (Government of the State of Munich, Upper Bavaria, Germany).

[0080] Screening of cyclodextrin-containing buffers for cholesterol extraction Cholesterol extraction was measured using the Cholesterol / Cholesterol Ester-Glo™ Assay (Promega, Madison, USA). 25 mg of PFA-fixed mouse liver was soaked in 1% w / v of different cyclodextrin-containing buffers: 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 MKBZ6644V), triacetyl-β-cyclodextrin (Sigma-Aldrich, 332623-10G, LOT STBJ9765), succinyl-β-cyclodextrin (Sigma-Aldrich, 85990-500MG, LOT The samples were incubated in 3 ml of heptakis(2,6-di-O-methyl)-β-cyclodextrin (Sigma-Aldrich, 39915-1G, LOT BCCF6041), γ-cyclodextrin (Sigma-Aldrich, C4892-5G, LOT SLBJ8855V), and α-cyclodextrin (Sigma-Aldrich, 779008-100G, LOT BCCJ0084). Assays were performed at different time points (days 2, 3, 5, and 7). A 5 μl aliquot of the supernatant was diluted 10-fold with cholesterol dissolving solution and incubated at 37°C for 30 min. Cholesterol detection reagent was then added to the samples and incubated at room temperature for 60 min. Values ​​were measured using a Centro LB 96 plate-reading luminometer (Berthold, Bad Wildbad, Germany).

[0081] Evaluating the effect of different cyclodextrin-containing buffers on antibody stabilization The homogeneity of antibodies in different cyclodextrin-containing buffers, i.e., the homogeneity of antibody aggregation, was measured by dynamic light scattering (DLS). A TH primary antibody was selected to evaluate antibody stabilization and homogeneity. TH antibody (Millipore, AB152) (MW: 150 kDa, concentration: 10 g / L) was dissolved in buffer with 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 was diluted and then measured in a folded capillary cell (DTS 1070) using a Zetasizer Nano ZS (Malvern, Worcestershire, UK). Samples were measured in triplicate with six subruns each. The temperature was set at 25°C.

[0082] Permeabilization function of different cyclodextrin-containing buffers on brain halves using methylene blue Half mouse brains were incubated with 45 mL of 1% (w / v) various cyclodextrin buffers at 37°C for 3 days. After two washes with PBS, 45 mL of 0.03% methylene blue was added to the samples and incubated overnight at 37°C. To determine the efficiency of methylene blue staining after incubation with the different CD buffers, the samples were cut in half at the midline to assess the efficacy of internal tissue staining. Camera images of the samples were analyzed by ImageJ along a profile plot, and pixels were quantified under a threshold gray value.

[0083] Perfusion and whole-body mouse fixation Mice were deeply anesthetized (intraperitoneally with 0.05 mg / kg fentanyl, 0.5 mg / kg medetomidine, and 5 mg / kg midazolam) and intracardially perfused with heparinized 0.01 M PBS (10–25 U / ml final heparin concentration, Ratiopharm, N68542.03; ISMATEC peristaltic pump system, perfusion volume 12 ml / min). After flushing the blood from the mice for 5–10 min, they were perfused with 4% paraformaldehyde (PFA) in 0.01 M PBS (Morphisto, 11762.01000) for 10–20 min. The skin of the mouse body was peeled off and postfixed in 4% PFA for 6 h at 4 °C before being transferred to 0.01 M PBS.

[0084] wildDISCO whole body immunostaining, PI labeling and tissue clearing The wildDISCO whole-body immunostaining protocol is primarily based on a setup for whole-body perfusion by pumping the pretreatment solution and immunostaining buffer through the mouse heart and vasculature. The pump setup has been previously described (Cai, R. et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nature Neuroscience 22, 317-327 (2019) and Zhao, S. et al. Cellular and Molecular Probing of Intact Human Organs. Cell 180, 796-812.e719 (2020)). Briefly, after PFA post-fixation and two 30-minute washes with 0.1 M PBS, the mouse was placed in a 300 ml glass chamber, and a perfusion needle was inserted into the mouse heart through the same hole as the PFA perfusion. The perfusion needle was then connected to an ISMATEC peristaltic pump (REGLO Digital MS-4 / 8 ISM 834; reference tubing, SC0266), which maintained a pressure of 160–230 mmHg (45–60 rpm) and was used to establish transcardial circulation. The pump had two channels. The first channel was used to pump the solution through the heart to circulate throughout the mouse, while the second channel collected and circulated the solution exiting the mouse's body. In the first channel, a 1 ml syringe tip (Braun, 9166017 V) was used to connect the perfusion needle (Leica, 39471024) to the reference tubing (Ismatec Reglo, SC0266) from the pump, which was set up to circulate the solution through the heart and into the vascular system. The second channel recirculated the solution, so the inlet tubing was immersed in the solution chamber of the glass chamber. After the pump and channels were set up, the needle tips were fixed with strong adhesive (Pattex, PSK1C) to ensure continuous and stable perfusion. All of the following perfusion steps were performed using the above setup.

[0085] Mice were first perfused overnight with 0.1 M PBS at room temperature, followed by 2 days of decalcification with a decalcification solution containing 10% w / v EDTA (Carl Roth, 1702922685) in 0.1 M PBS. The pH was adjusted to 8–9 with sodium hydroxide (Sigma-Aldrich, 71687) at room temperature to decalcify all bones. The mice's bodies were then perfused three times with 0.1 M PBS, each time for 3 hours. Next, the mice were perfused for 1 day with a permeabilization and blocking solution containing 10% goat serum and 2% Triton X-100 in 0.1 M PBS. 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, 1:10,000) or 290 μl of PI (stock concentration 1 mg / ml) incubated in 250 ml of immunostaining buffer containing 3% goat serum, 10% CHAPS, 2% Triton X-100, 10% DMSO, 1% glycine, and 1% heptakis(2,6-di-O-methyl)-β-cyclodextrin in 0.1 M PBS. The mouse bodies were then washed three times in 0.1 M PBS, each time for 12 hours at room temperature. The mouse bodies were then incubated for 7 days in immunostaining buffer at room temperature with Alexa Fluor 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, 1:10,000 dilution). The mouse bodies were washed three times in 0.1 M PBS, each time for 12 hours.

[0086] After immunostaining, the mice were transferred to a fume hood and cleared using the 3DISCO passive whole-body clearing protocol described in Ku, T. et al. Elasticizing tissues for reversible shape transformation and accelerated molecular labeling. Nature Methods 17, 609-613 (2020). Briefly, the mouse body was placed in a 300 ml glass chamber and immersed in a 200 ml gradient of THF (tetrahydrofuran, Roth, CP82.1) in distilled water (50% x 1, 70% x 1, 80% x 1, 100% x 2, 12 h for each step) with gentle shaking. The mice were then immersed in dichloromethane (DCM, Sigma, 270997) for 3 h and finally in BABB solution (benzyl alcohol + benzyl benzoate 1:2, Sigma, 24122 and W213802) until the body became optically transparent.

[0087] Light sheet microscope imaging Image stacks were acquired using a Blaze Ultramicroscope (LaVision BioTec GmbH, version 7.3.2) equipped 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. Mouse whole bodies were individually scanned with a Blaze Ultramicroscope light-sheet microscope 4x objective (Olympus XLFLUOR 4x corrected / 0.28 NA [WD=10 mm]). We imaged the entire mouse with 9x23 tile scans covering the entire mouse with 20% overlap, separated from the ventral and dorsal surfaces to a depth of 10 mm, with a Z-step of 10 μm to cover the whole body volume. 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 intensity of the fluorescent signal to avoid saturation. The acquired raw image TIFFs were processed with the Fiji stitch plugin (http: / / www.discotechnologies.org / ).

[0088] Virtual reality (VR) headset operation Images require a virtual reality (VR) headset. To visualize them, you need a VR device or a VR movie player on your computer. Images played in virtual reality must have "_360" at the end of their filename and be set to "360° / 3D" view in the VR player for an immersive experience.

[0089] Reconstruction and quantification of whole-body scans Detailed step-by-step instructions for image data stitching and volume fusion have been provided previously (Zhao, S. et al. Cellular and Molecular Probing of Intact Human Organs. Cell 180, 796-812.e719 (2020)). Briefly, image stacks were recorded using ImSpector software (LaVision BioTec GmbH) and saved in TIFF format for each channel separately. Scanned ventral and dorsal mouse image data were first stitched using the Fiji Stitch plugin, and volumes were fused using Vision4D (v3.5 x64, Arivis AG, version 3.4.0). Alignment was performed by manually selecting three to four anatomical landmarks from the overlapping region to increase the accuracy of volume fusion. Representative images were created using Imaris (Bitplane AG, version 9.6.0) and Vision4D for 3D volume reconstruction, maximum intensity projection, and depth color rendering. The Imaris surface tool was manually used to isolate specific tissue regions, and the mask channel option for pseudocoloring was selected. After manual segmentation, the region was visualized in 2D slices using the Ortho Slicer tool. Virtual reality (VR) photographs and images were generated using Syglass software (IstoVisio, version 1.7.2). To quantify the myenteric plexus in the duodenum between germ-free and wild-type mice, five 200 μm × 200 μm × 200 μm cubic volumes along the portal triad were randomly selected from the reconstructed 3D image in Imaris. The length of the PGP9.5-positive myenteric plexus in each volume was traced using the Imaris Filament Tracer.

[0090] Quantification Data are presented as mean ± SD. Statistical analysis was performed using Prism GraphPad software version 8 with a 95% confidence interval. P values ​​were calculated using an unpaired, two-tailed t-test to compare data between two groups. A P value of <0.05 was considered statistically significant. [Example]

[0091] Cholesterol extraction; methylene blue penetration into whole mouse brain; prevention of antibody aggregate formation We investigated the potential function of β-cyclodextrin variants (Fig. 1a) with various R motifs (e.g., methyl, hydroxypropyl, hydroxyethyl, succinyl, and acetyl) in enhancing cholesterol extraction and permeabilization in fixed samples using CHAPS and Triton X-100 detergents (Fig. 3a). Using a cholesterol / cholesterol ester-glo assay to evaluate cholesterol extraction, we found that heptakis(2,6-di-O-methyl)-β-cyclodextrin (CD5) extracted most of the cholesterol from mouse liver tissue after 7 days (Fig. 1b). CD2 (2-hydroxypropyl-β-cyclodextrin) also exhibited good cholesterol extraction function. Addition of CD5 to the permeabilization reaction resulted in rapid and uniform penetration of methylene blue throughout the mouse brain within 12 hours (FIG. 1c and FIG. 3b). Cyclodextrins stabilize proteins in solution by preventing aggregation, as reported in Serno, T., Geidobler, R. & Winter, G. Protein stabilization by cyclodextrins in the liquid and dried state. Advanced Drug Delivery Reviews 63, 1086-1106 (2011). Therefore, we measured the antibody size in the antibody solution using dynamic light scattering (DLS). After 7 days at room temperature without a CD5-containing solution, the antibody exhibited two peaks in the DLS data: one at 11.5 nm, presumably corresponding to the antibody monomer, and the other at a larger size, most likely corresponding to a different aggregation state. Addition of CD5 prevented aggregate formation (Figure 1d). [Example]

[0092] Increased uniformity and depth of antibody staining throughout the mouse body We next tested whether the enhanced membrane permeabilization and reduced aggregation tendency of antibodies in CD5-containing buffers could enhance the uniformity and depth of antibody staining throughout the mouse body. Different nervous systems, such as the sympathetic and parasympathetic nervous systems, regulate and coordinate organ function. To reveal the delicate innervation of organs throughout the mouse body, we stained the peripheral nerve network in juvenile adult mouse bodies (approximately 4 weeks old, approximately 10 × 3 × 2 cm in size) with protein gene product 9.5 (PGP9.5), a pan-neuronal marker (Figures 1e and 4a). After labeling the whole mouse antibodies using CD5-containing buffer, the body was made optically transparent and pan-optic imaging was performed using a light-sheet microscope. The peripheral nervous system was stained uniformly, without systematic differences in signal intensity, among different tissues such as vertebrae (FIG. 1f) and adipose tissue (FIG. 1g), and at different depths in the mouse body.

[0093] In the heart, for example, a network of nerve fibers running through the ventricular myocardium was evident (Figure 4b). The splenic parenchyma displayed a complex spike-like structure of nerve fibers (Figure 4c). The vagus nerve branched into smaller fiber bundles as it progressed toward the dorsal spleen, and we also visualized the splenic nerve network. PGP9.5+ nerve fibers also innervated the hepatic sinusoids (Figure 4d) and were distributed end-to-end along the hepatic duct. In the gallbladder, a ganglionic plexus containing a series of irregularly shaped ganglia (Figure 4d) was clearly visible. In the small intestine, an interconnected ganglionic plexus was observed on the intestinal wall (Figure 4e).

[0094] In the 3D reconstruction of the scans, we were able to easily observe the nerves innervating various organs. For example, we were able to track the vagus nerve, which provides parasympathetic innervation to abdominal organs connecting internal organs such as the kidneys, adrenal glands, ureters, liver, spleen, and gastrointestinal (GI) tract (Figure 1h and Figure 4f), a task that was greatly facilitated using virtual reality visualization techniques. Compared to whole-organ antibody staining, whole-mouse tracking allowed us to visualize the neural connections between different organs (Figure 1h). This provides essential clues for understanding the role of neurotransmission in normal physiology and disease. [Example]

[0095] Staining of lymphatic vessels and immune cells To demonstrate the generality of this approach, lymphatic vessels and immune cells were stained with lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and CD45, respectively.

[0096] At the whole-mouse level, the ultrastructural lymphatic network was observed throughout each body segment (Figure 1i), allowing detailed visualization of lymphatic organization in individual organs. For example, LYVE-1+ blood vessels were observed in the liver sinusoidal endothelium and superficial gastrocnemius muscle (Figures 5a and 5b). LYVE-1+ lymph nodes were observed near the hind limbs. LYVE-1+ cells with distinct shapes were particularly evident in adipose tissue (Figure 5c). Larger lymphatic vessels in the kidney branched into lymphatic capillaries with a dendritic structure (Figure 5d). Tracheal lymphatic vessels displayed a segmental pattern of interconnected vessels (Figure 5e). In the stomach, lymphatic vessels were distributed unevenly throughout the stomach wall and branched in a dendritic pattern (Figure 5f). Blunt-ended tubular lymphatic vessels (ductules)20 were clearly located in the intestinal villi, and abundant and well-organized lymphatic plexuses and networks were observed on the outer surface of the intestinal wall (Figures 5g–5i). Although it has traditionally been proposed that the brain parenchyma lacks lymphatic vessels (Cugurra, A. et al. Skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and CNS parenchyma. Science 373, eabf7844 (2021) and Louveau, A. et al. Structural and functional features of central nervous system lymphatic vessels. Nature 523, 337-341 (2015)), lymphatic drainage from the CNS via meningeal lymphatic vessels exists. Our whole-body immunolabeling data showed small, short lymphatic vessels entering the brain parenchyma from the meninges. Several LYVE-1+ lymphatic vessels were also observed connecting the olfactory bulb and cortex (Figure 1j), as observed by LYVE1 and PROX1 staining, respectively. We also found lymphatic vessels entering the brain parenchyma around the thalamus (Figure 1k), which was confirmed by both LYVE1 and podoplanin staining. [Example]

[0097] Studying the relationship between different physiological systems in the same mouse The compatibility of wildDISCO with previously validated antibodies for labeling allowed us to study the relationship between different physiological systems in the same mouse. First, we co-immunolabeled tyrosine hydroxylase (TH)+ sympathetic nerves and CD45+ immune cells (Figures 2a-2c, 6). Substantial co-localization of immune cells was found along the vagus nerve, particularly in the inferior mesenteric plexus (Figures 2a and 2b), and at frequent contacts between immune cells and sympathetic nerves on the intestinal wall (Figure 2c).

[0098] To better demonstrate neuroimmune interactions in the lymphatic system, particularly in lymph nodes (LNs), we used double staining of nerve fibers and lymphatic vessels (Figure 2d, Figures 7-8). Notably, large LYVE1+ (Figure 2e) and PROX1+ (Prospero homeobox protein 1, a marker of lymphatic endothelium) (Figure 2g) LNs were detected in the mouse hindlimbs (Figure 2e, Figure 2g). CD45 staining confirmed that the observed structures were LNs (Figure 2f). Co-staining with the pan-neuronal marker PGP9.5 or the peripheral sympathetic neuron marker TH demonstrated neuronal processes innervating the LNs.

[0099] Next, we evaluated the effects of biological perturbations using cultured wildDISCO. To do so, we compared the structure of the gut-associated nervous system in germ-free mice with that in specific pathogen-free (SPF) control mice. Double staining data for lymphatic and immune cell-associated nerves already demonstrated the intricate details of the enteric nervous system throughout the gut (Figures 2a-2d). When studying germ-free mice, we found that the PGP9.5+ neural lattice network was substantially less dense compared to wild-type mice. The density of the myenteric plexus was 1.478 (×10 3 mm / mm 3 ) to 0.659(×10 3 mm / mm 3 ) (Figure 2h), confirming the importance of gut microbiota interactions in the development and / or maintenance of the mesenteric plexus (Fuelling, C., Dinan, TG & Cryan, JF Gut Microbe to Brain Signaling: What Happens in Vagus. Neuron 101, 998-1002 (2019)). [Example]

[0100] wildDISCO applied to a cancer metastasis model Tyrosine hydroxylase (TH) was co-stained with breast cancer cells MDA-MB-231. Cancer cells were clearly visible in close proximity to TH+ nerves. The results are shown in Figure 10. [Example]

[0101] wildDISCO reveals the entire arterial network in mice Alpha-smooth muscle actin (alpha-SMA) was applied to examine arterial distribution on a whole-body scale, and the results are shown in Figure 11. [Example]

[0102] Further antibody validation To demonstrate the broad applicability of this approach, the uniformity and depth of antibody staining was confirmed with additional antibodies using the methods described above. The list of antibodies tested is shown in Tables 2 and 3 below.

[0103] Table 2. Verified antibodies

[0104] [Table 2] Working / partially working Table 3. Further validated antibodies

[0105] [Table 3] [Example]

[0106] Comparison of different clearing methods for mouse whole-body antibody staining As shown by the results in Figures 13 and 14, wildDISCO shows superior staining throughout the mouse body compared to other clearing techniques such as vDISCO, iDISCO, uDISCO, and the PEGASOS method. In particular, wildDISCO was evaluated against other established methods (vDISCO, iDISCO, uDISCO, and PEGASOS) for their efficacy in staining whole mice with standard antibodies. In a direct side-by-side comparison using synapsin 1, incomplete staining was observed in iDISCO, uDISCO, and PEGASOS-stained samples, along with blurred imaging of many organs (e.g., the head and hind limbs) (Figure 13a, b). For example, in contrast to vDISCO, wildDISCO was able to completely penetrate the antibody uniformly and deeply throughout mouse internal organs, including the hind limbs, spine, forelimbs, kidneys, and liver (Figure 13b, c and Figure 14).

[0107] Materials and Methods : The detailed protocol for vDISCO is described in Cai, R. et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nature Neuroscience 22, 317-327 (2019). The nanoboosters used here were replaced with commercially available IgG antibodies at a concentration of 25 μg per mouse, as shown in Table 4 below. Table 4. Protocols and buffers used for vDISCO and wildDISCO

[0108] [Table 4] For the iDISCO antibody staining method, we followed the latest protocol update from the iDISCO+ publication Renier, N. et al. iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging. Cell 159, 896-910 (2014) and https: / / iDISCO.info. Briefly, each step was adjusted to the whole-body level rather than the organ level (e.g., the duration of methanol dehydration was extended to 6 hours). After pretreatment with methanol, the whole body was treated with a series of iDISCO+ solutions. For immunolabeling, whole mice were treated with primary antibody (synapsin-1, CST, no. 5297, 25 μg, 1:10,000 dilution in 250 ml of PBS containing 0.2% Tween-20, 5% DMSO, 3% donkey serum, and 1 mg / L heparin) for 7 days at 37°C, followed by incubation with secondary antibody (Alexa Fluor 647 goat anti-rabbit IgG, Thermo Fisher Scientific A-21245, 25 μg, 1:10,000 dilution in 250 ml of PBS containing 0.2% Tween-20, 5% DMSO, and 3% donkey serum) for 7 days at 37°C. Finally, the whole mice were washed until optically clear.

[0109] For uDISCO and PEGASOS whole-mouse labeling, the immunostaining buffer and process were adapted from the iDISCO method described above. The uDISCO and PEGASOS clearing steps were then performed based on the publications Pan, C., Cai, R., Quacquarelli, F.P., Ghasemigharagoz, A., Lourbopoulos, A., Matryba, P., Plesnila, N., Dichgans, M., Hellal, F., and Erturk, A. (2016). Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods and Jing, D. et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 28, 803-818 (2018). [Example]

[0110] Visualization and analysis of tumor-associated TLS in tumor metastasis models wildDISCO is uniquely able to interrogate tertiary lymphoid structures (TLS) and their distribution in the whole mouse, as demonstrated by the results in Figure 15. Specifically, to visualize cancer metastasis throughout the body, BALB / c mice were subcutaneously implanted with 4T1 breast cancer cells, as described in detail below. Conventional B cells, follicular dendritic cells, and T cell clusters and / or aggregates (representing TLS) were found in the primary tumor (Fig. 15c-h) and in some metastatic sites (e.g., lung and intestinal metastatic sites) (Fig. 15b, i-l).

[0111] Materials and Methods: 4T1 breast cancer cells encoded with EGFP and enhanced firefly luciferase were filtered through a 100 μm membrane and resuspended in RPMI1640 medium (GIBCO, 11875093). For the subcutaneous injection model, 1 × 10 6Cancer 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, metastases in mice were measured by bioluminescence using an IVIS Lumina II Imaging System (Caliper Life Sciences). Briefly, mice were anesthetized with ketamine, secured in an imaging chamber, and imaged 15 minutes after luciferin injection (150 mg / kg, intraperitoneal injection). Bioluminescence signals were quantified using Living Image Software v.4.2 (Caliper Life Sciences). After confirming metastatic development by IVIS imaging, mice were euthanized and stained with two antibodies, CD3 and CD23, to confirm TLS. CD5 (heptakis(2,6-di-O-methyl)-β-cyclodextrin) was used in this experiment. Labeling solution (see the table above, including wildDISCO buffer in Example 9) was used. The clearing solution was THF, DCM, and BABB. In summary, a novel DISCO technique ("wildDISCO") is provided that enables high-resolution 3D imaging of the peripheral nervous system (PNS), lymphatic system, and vasculature in the whole body of mice. This technique can generate comprehensive biological maps of nerves, blood vessels, and lymphatic vessels using conventional antibodies for whole-body staining in mice. It reveals pathological changes such as tertiary lymphatic structures in cancer, enables precise tracking of therapeutic molecules and cells, and enhances our understanding of disease pathology and treatment.

[0112] Most diseases involve multiple interconnected physiological systems, yet histological assessment of their pathology is currently limited to small tissue samples. Herein, we describe wildDISCO, a technique involving cholesterol extraction to allow deep tissue penetration of large fluorescent dye-containing labeling agents, such as a standard 150 kDa IgG antibody, in chemically fixed whole mice. By combining wildDISCO with whole-mouse clearing, we created a full-body map of the nervous, immune, and lymphatic systems, demonstrating their intimate interactions throughout the mouse body.

[0113] For example, wildDISCO can reveal integrated neural, vascular, and lymphatic networks. Using this technique, we were able to observe PNS innervation in most organs, including the heart, lungs, liver, kidneys, stomach, and intestine. Furthermore, the vagus nerve, which innervates the gastrointestinal tract, was visualized. Using this technique, we also demonstrated lymphatic capillaries that heterogeneously permeate the center of the intestinal villi and region-specific 3D villous lymphatic networks. Surprisingly, we found that lymph nodes are innervated by a population of PNs with immunoregulatory capabilities. Using this technique, we also imaged organ-specific vascular patterns and transcortical capillary networks as the primary support for multiple bones. Therefore, mapping the mouse whole-body system can provide a roadmap for diverse studies, including neural circuits, immune regulation, and angiogenesis in the mammalian body.

[0114] Our method allows for unbiased imaging of the entire mouse body 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, as it allows for the use of pre-made IgG antibodies to uniformly and simultaneously stain structures throughout the mouse body. The mouse head, combining hard tissue (skull) and soft tissue (brain), is a perfect example of the versatility of this method. Using wildDISCO, we can map all lymphatic vessels within and around the brain parenchyma of an intact mouse head.

[0115] The wildDISCO technique achieves uniform and simultaneous antibody staining throughout the mouse body, enabling previously unavailable 3D anatomical information (e.g., aided by VR visualization) to provide a more comprehensive understanding of the initiation, progression, and extent of pathology at the whole-organism level in mice.

[0116] Industrial Applicability The methods and products of the present invention are industrially applicable and can be used, for example, to test biopharmaceuticals such as therapeutic antibodies.

[0117] References

[0118] Table 5 TIFF2025536457000009.tif217170

Claims

1. 1. A method for preparing animal tissue for fluorescence microscopy, comprising:

1. A method comprising: labeling a target molecule in a fixed animal tissue with a labeling solution comprising a fluorescent dye-containing labeling agent capable of binding to the target molecule to obtain fixed animal tissue labeled with the fluorescent dye-containing labeling agent, wherein the molecular weight of the fluorescent dye-containing labeling agent is greater than 100 kDa; wherein the fixed animal tissue is treated with a permeabilization solution, and the labeling solution comprises a cyclodextrin derivative.

2. Cyclodextrin derivatives include those having the following chemical formula: 【Chemistry 1】 (In the formula, m is 6 to 8; R ２ , R ３ and R ６ are each independently substituted by H and optionally one or more groups selected from OH, oxo, and COOH; １ ~C ６ alkyl; Non-hydrogen groups R per glucopyranose unit ２ , R ３ and R ６ The degree of substitution (DS), which represents the average number of ２ , R ３ and R ６ are H and CH ３ When selected from the group consisting of The method of claim 1 , wherein the structure is:

3. 3. The method of claim 2, wherein m is 7.

4. (a) R ２ and R ６ is a straight chain or branched C １ ~C ６ alkyl, and R ３ is H, or (b) R ２ and R ３ is a straight chain or branched C １ ~C ６ alkyl, and R ６ is H, or (c) R ３ and R ６ is a straight chain or branched C １ ~C ６ alkyl, and R ２ is H, The method according to claim 2 or 3.

5. R ２ and R ６ is CH ３ and R ３ The method of any one of claims 2 to 4, wherein is H.

6. The method according to any one of claims 1 to 5, wherein the cyclodextrin derivative is heptakis(2,6-di-O-methyl)-β-cyclodextrin.

7. The method according to any one of claims 1 to 6, wherein the fluorescent dye-containing labeling agent is an antibody conjugated to a fluorescent dye, the antibody being capable of binding to a target molecule.

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

9. Before labeling the target molecules in the fixed animal tissue with the labeling solution, the following steps are performed: A method comprising contacting the fixed animal tissue with a primary antibody capable of binding to a structure or protein, lipid, DNA or RNA present in the fixed animal tissue, or contacting the fixed animal tissue with a primary antibody capable of binding to a protein present in the fixed animal tissue, wherein the fluorescent dye-containing labeling agent is capable of binding to the primary antibody.

10. Before labeling the target molecules in the fixed animal tissue with the labeling solution, the following steps are performed: and treating the fixed animal tissue with a blocking solution to block non-specific antigen binding of the antibody, wherein: The method of any one of claims 1 to 9, wherein the blocking solution comprises animal serum.

11. below: removing heme from the fixed animal tissue, and treating the tissue with a permeabilization and blocking solution to decolorize, permeabilize, and block the tissue; and 11. The method according to claim 1, comprising, in this order, labeling a target molecule in the fixed animal tissue with a labeling solution comprising a cyclodextrin derivative and a fluorescent dye-containing labeling agent capable of binding to the target molecule to obtain fixed animal tissue labeled with the fluorescent dye-containing labeling agent, wherein the molecular weight of the fluorescent dye-containing labeling agent exceeds 100 kDa.

12. The method according to any one of claims 1 to 11, wherein the whole body of the animal is subjected to labeling.

13. Use of a cyclodextrin derivative according to any one of claims 2 to 6 for improving labelling of target molecules in fixed animal tissues or whole animals with fluorescent dye-containing labelling agents.

14. (a) R ２ and R ６ is a straight chain or branched C １ ~C ６ alkyl, and R ３ is H, or (b) R ２ and R ３ is a straight chain or branched C １ ~C ６ alkyl, and R ６ is H, or (c) R ３ and R ６ is a straight or branched chain C １ ~C ６ alkyl, and R ２ is H, 14. The use according to claim 13.

15. R ２ and R ６ is CH ３ and R ３ The use according to claim 13 or 14, wherein is 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 fluorescent dye-containing labeling agent exceeds 100 kDa, and the fluorescent dye-containing labeling agent is an antibody conjugated to a fluorescent dye, wherein the antibody is capable of binding to a target molecule.

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

19. The use according to any one of claims 13 to 18, wherein the whole body of the animal is subjected to labeling.

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

21. (a) R ２ and R ６ is a straight chain or branched C １ ~C ６ alkyl, and R ３ is H, or (b) R ２ and R ３ is a straight chain or branched C １ ~C ６ alkyl, and R ６ is H, or (c) R ３ and R ６ is a straight or branched chain C １ ~C ６ alkyl, and R ２ is H, 21. The composition of claim 20.

22. R ２ and R ６ is CH ３ and R ３ 22. The composition of claim 20 or 21, wherein is H.

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

24. The composition of any one of claims 20 to 23, further comprising a zwitterionic surfactant.

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

26. 26. The composition of 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. Additionally, in an aqueous buffer solution, the following components: animal serum, organic solvents, and / or amino acid, 28. The composition of any one of claims 24 to 27, comprising one or more or all of:

29. Additionally, in phosphate buffered saline (PBS), the following components: mammalian serum, a water-miscible organic solvent, and glycine, 28. The composition of any one of claims 24 to 27, comprising one or more or all of:

30. When present, the following concentrations of said ingredients: the cyclodextrin derivative is 0.5-2, 0.75-1.5, or 1% w / v; the animal serum is 0.5-12, 0.75-10, or 1-3% v / v; the zwitterionic detergent is 5-15, 7.5-12.5, or 10% w / v; the non-ionic surfactant is 0.5-4, 1-3, or 2% w / v; the organic solvent is 5-20, 7.5-15, or 10% w / v; the amino acid is 0.5-2, 0.75-1.5, or 1% w / v; the buffer concentration of the aqueous buffer is 0.05-0.2M, 0.08-1.2M, or 0.1M; 30. The composition of claim 28 or 29.

31. The composition according to any one of claims 20 to 30, having a pH value of 7 to 7.4 or 7.2.

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