Chaotrope-assisted deep immunostaining

Abstract

A supramolecular histochemistry system for staining includes a chaotropic ion and a chaotropic ion complexing agent that acts as a molecular host and accepts the chaotropic ion as a molecular guest. The chaotropic ion facilitates the diffusion of a probe, such as an antibody, into and within a tissue sample, binding the probe to the molecular host or diluting the probe, thereby promoting the association of the antibody with a target antigen and generating an immunostaining or histochemistry signal. Alternatively, a small fluorescent probe serves as the molecular guest and is complexed by the supramolecular host to facilitate deep penetration. A method for performing histology employs the supramolecular system with deep, uniform histochemistry. Such a method is relatively rapid, scalable, automatable, and cost-effective.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 63 / 387,773, filed December 16, 2022, which is incorporated by reference in its entirety, including any tables, figures, or drawings. [Background technology]

[0002] In pathology and biomedical research, there is a clinically unmet need for 3D observation of tissue specimens to better stratify the risk of disease progression and detect ominous features such as the amount of involved margin and the presence of high-risk molecular markers. 3D histological methods must be scalable at a reasonable cost to be translated into clinical procedures. The methods must be scalable in terms of tissue size, tissue processing time, and throughput of the number of specimens that can be processed per day, and must be able to produce reliable results despite variations in specimen quality. Thus, tissue-based diagnosis and prognosis can become practical and reliable.

[0003] Current methods for analyzing the distribution and localization of specific proteins within individual cells or tissues are performed using immunostaining. Immunostaining is a standard technique that uses an antibody that targets a specific molecule, i.e., a primary antibody, to detect its presence. A secondary antibody conjugated with a fluorescent or enzyme label binds to the primary antibody, thereby allowing visualization and / or quantification using a fluorescence microscope or by adding a chromogenic substrate. The quality of the staining depends on the specificity and quality of the primary antibody.

[0004] Antibodies used for immunostaining can have difficulty penetrating cells and tissues, and the cellular uptake of impermeable molecules presents challenges for drug discovery and diagnostic purposes. The mechanistic reasons for poor antibody penetration in large tissue blocks are unclear, but it is thought to be related to a reaction-diffusion process. Briefly, antibodies are relatively free to move within permeabilized and fully delipidated tissues. However, their reaction with target antigens or binding partners (via nonspecific electrostatic interactions) can cause their deposition at the surface of the tissue block, depleting the antibody and preventing its deep penetration. Worse yet, this can lead to nonuniform staining, resulting in a bright signal rim formed only at the surface while deep penetration into the tissue is not observed.

[0005] Research to improve 3D observation of tissue specimens has focused on tissue clearing, and several clearing agents are now commercially available for research use. 3D tissue staining is equally applicable to staining large tissue blocks followed by serial sectioning, allowing for the convenient production of prestained slices, which is particularly useful for tomography techniques. This emerging trend in 3D tissue imaging is paralleled by the development of novel probes, specialized optical objectives, and new programs for visualizing and analyzing very large data sets.

[0006] Current immunostaining techniques for 3D histology used in clinical specimens and tissue diagnosis include eFLASH, ELAST, CUBIC-Histo Vision, SPEARS-THICK staining, and intravascular antibody perfusion.

[0007] eFLASH is an electrochemical method that uses the surfactant sodium deoxycholate at a certain pH to reduce antibody trapping by surface antigens and an electric field to enhance antibody mobility, thereby promoting deep antibody diffusion. eFLASH has been successfully applied to deep, uniform, and quantitative immunostaining of whole mouse or marmoset brains and is applicable to inexpensive, commercially available antibodies. Dedicated equipment, while reusable, is expensive, and its scalability is limited by the maximum sample size and the number of samples that can be processed simultaneously.

[0008] ELAST is based on the conversion of tissue into an elastic material by embedding it in situ in long-chain polyacrylamide. When compressed, the diffusion distance required by macromolecular probes is significantly shortened, facilitating diffusion and deep penetration. In practice, a dedicated automated machine is required for continuous compression and relaxation, and considerable labor is required to peel off excess polyacrylamide gel and manually load different sizes of tissue gel into the compressor. Furthermore, the tissue becomes fragile, and its morphology and structure become distorted within the tissue.

[0009] CUBIC-HistoVision (Cubic-HV) uses chemical buffers to adjust the gel-electrolyte properties of tissues to achieve staining in thick tissues, including successful staining of whole mouse brains with multiple antibodies. Unfortunately, incubation after complete tissue delipidation takes a very long time and requires large amounts of antibodies.

[0010] SPEARs are heat-stabilized antibodies for heat-promoted deep antibody penetration in thermal immunohistochemistry (ThICK-staining) with optimized kinetics. Although the method's turnaround time is scalable, the signal intensity is weak, often resulting in false-negative detection of the antigen, and heat stabilization can result in nonspecific staining.

[0011] Intravascular antibody perfusion works by using the bulk of the body's fluid flow to deliver the probe directly through the patient's blood vessels. The antibody then diffuses a short distance from the blood vessels to the nearest antigen. It assumes that the antigen is easily labeled and that the vasculature is uniformly distributed within the tissue. However, this method requires vast amounts of antibody due to the very large perfusion rates, and it is not scalable because not all tissues have easily cannulated blood vessels.

[0012] Therefore, there is a need for improved histological systems for two- and three-dimensional analysis of tissue specimens, including novel in situ supramolecular methods that simultaneously improve the penetration depth and uniformity of immunostaining in thick biological tissue specimens.

[0013] [Summary of the Invention] An embodiment of the present invention relates to a supramolecular system for immunostaining. The system includes at least one chaotropic ion for improving the penetration and diffusion of at least one probe into tissue, where the probe may be an antibody, such as, but not limited to, a fluorescently labeled antibody, other protein, or small molecule in a tissue sample. Optionally, the system may further include at least one complexing agent that acts as a molecular host and accepts the chaotropic ion as a molecular guest, promoting the association of the probe, e.g., an antibody, with a target antigen, thereby generating a signal at the end of the experiment.

[0014] The chaotropic ion may be a perrhenate ion, a closo-borane ion, a closo-carborane ion, or a Keggin-type polyoxometalate of tungsten, molybdenum, tantalum, niobium, or vanadium. The chaotropic ion may be derived from an unsubstituted closo-borane of more than nine atoms, a closo-carborane of more than nine atoms, or a mixture thereof, where the boron or carbon atoms of the cage structure are independently substituted with hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, amino, or alkyl groups. An exemplary chaotropic ion is closo-dodecaborate.

[0015] The complexing agent may be or have the functionality of a cyclodextrin, cucurbituril, calixarene, cyclophane, cryptand, cryptophane, or chemical derivatives thereof, or an oligomer having anion-associating repeating units. Exemplary complexing agents include hexakis-β-glucopyranose (α-cyclodextrin), heptakis-β-glucopyranose (β-cyclodextrin), and octakis-β-glucopyranose (γ-cyclodextrin). wherein one or more of the hydroxy groups are independently optionally substituted with an amino group, a methoxy group, a (2-hydroxy)propoxy group, a sulfato group, a guanidino group, a phosphat group, an acetamido group, an azido group, a bromo group, an iodo group, a chloro group, a toluenesulfonyl group, a thiol group, a succinyl group, a phosphat group, a 4-sulfatobutoxy group, a carboxymethoxy group, or a (2-aminoethyl)amino group. In some embodiments, the complexing agent may be omitted if the effect of closo-dodecaborate, or other chaotropic ions, is simply diluted by water or any chemical buffer system.

[0016] The supramolecular system may use a chemical buffer system, including, but not limited to, Good's buffer, phosphate-buffered saline (PBS), Tris, CAPS, HEPES, or any combination thereof. The buffer system may have a pH of 6-11 and may be present at a concentration of 10-1000 mM. For example, 1x PBS with a pH of 7-8 may be used. The chaotropic ion and the chaotropic ion complexing agent may each be contained in a separate vehicle for separate introduction into the tissue sample.

[0017] One embodiment of the present invention relates to a method for performing two-dimensional or three-dimensional histology, in which the above-described supramolecular system for immunostaining is used with a specimen containing tissue, and the specimen is incubated with chaotropic ions of the supramolecular system to form an incubated specimen, which is combined with a complexing agent for the chaotropic ions of the supramolecular system to promote association of antibodies with target antigens and generate a signal.

[0018] The combining step may be performed as a step subsequent to the incubation step. The signal may be imaged, analyzed, and quantified. The tissue-containing specimen may be derived from a mammal, such as a mouse or a human, and may be used for experimental or clinical purposes. The tissue may be a whole organ or a portion thereof. The tissue-containing specimen may be a tissue slice having a thickness of about 1 to about 100 μm. The slice may be obtained by frozen sectioning, vibrotome sectioning, or paraffin sectioning, and may be fixed by formalin fixation, glutaraldehyde fixation, methanol fixation, ethanol fixation, glyoxal fixation, picric acid fixation, trichloroacetic acid fixation, polyacrylamide-formalin fixation, polyglycerol-3-polyglycidyl ether fixation, or a combination thereof. Optical tissue clearing and tomography may be used according to any known protocol, particularly in three-dimensional histology. [Brief explanation of the drawings]

[0019] [Figure 1A] FIG. 1 shows the chaotropic ion structures of closo-dodecahydro-dodecaborate and its derivatives. [Figure 1B] FIG. 1 shows the structures of various chaotropic ions. [Figure 2A] FIG. 1 shows the structure of a cyclodextrin complexing agent. [Figure 2B] FIG. 1 shows the structure of a complexing agent made of cyclodextrin and its derivatives. [Figure 3] 1 shows steps of a method according to one embodiment for deep staining using a supramolecular system (INSIGHT) according to one embodiment. The diagram is illustrated by the external view of the tissue (top) and the internal view of the tissue (bottom), suggesting that the interaction of components achieves the desired uniform deep staining. [Figure 4] Figure 1 shows an outline of the experimental design used to benchmark antibody penetration depth and uniformity using a mouse hemibrain, where the tissue is sectioned into planar channels after bulk staining, the sectioned channels are subjected to repeat staining, and the channels are analyzed before (bulk stain channel) and after (cut stain channel) the repeat staining. [Figure 5] Representative images from a specimen in an INSIGHT benchmark experiment are shown. In this figure, an XY-plane image is displayed for the bulk stain (top row), along with the corresponding calculated penetration length (top inset). These penetration lengths are relatively invariant for the same Z-depth (arranged in columns) of the images. An XY-plane image is also displayed for the section stain (bottom row), through the section plane (image plane). Here (bottom row), the penetration distance is uneven and non-uniform due to slight curvature of the tissue and irregular tissue structure. These features are taken into account when quantifying the penetration depth for the section stain (bottom inset). [Figure 6]FIG. 6 shows a plot of the ratio of bulk staining intensity to cleaved staining intensity for various γ-cyclodextrin derivatives obtained using a benchmark experiment, compared to the commonly used zwitterionic surfactant CHAPSO. [Figure 7] Figure 1 shows bulk staining images for INSIGHT and five existing imaging systems used for deep immunostaining. [Figure 8] 10 is a plot showing the relationship between cell segmentation and penetration distance for the entire image in bulk staining, compared to hypothetical ideal performance. [Figure 9] Figure 9 shows an xz projection of the white-boxed area in Figure 7, which corresponds to the area of ​​deep bulk stain penetration, with only the cut stain channel displayed. In this figure, it can be seen that the deeper the penetration, the more permeable the tissue. [Figure 10] Figure 10 shows plots of staining intensity versus penetration depth for cut staining to quantitatively compare tissue permeabilization by INSIGHT and five existing imaging systems. [Figure 11] This is a bar graph showing the number of antibodies that have been verified to be usable with INSIGHT and five existing imaging systems. [Figure 12] Segmented bar graph showing processing timelines for INSIGHT and five existing imaging systems, scaled based on the duration of each step, ignoring cleaning steps common to all systems that are of comparable magnitude. [Figure 13] Figure 1 shows a schematic diagram of a lateral flow rapid antigen test (RAT) kit used to verify the mechanism of action of supramolecular histochemistry as a reaction-diffusion model, and photographs of the RAT when different concentrations of [B12H12]2- are added to the sample pad. [Figure 14]Diagram showing the experimental steps (top) and the corresponding principle (bottom) in supramolecular histochemistry, in which tissue is infiltrated with a primary antibody, a fluorescently labeled secondary antibody Fab fragment, and a chaotropic ion (chaotropic agent), and then the tissue is transferred to a washing or dilution solution containing a chaotropic complexing agent. [Figure 15] FIG. 1 shows a nucleic acid probe (NAP) and its complexation with sulfobutylether-β-cyclodextrin (SBE-β-CD), which represents another supramolecular histochemical strategy to enhance the tissue penetration of the probe. [Figure 16] 1 is a graph showing the enhanced penetration depth of [DAPI⊂SBE-β-CD] in a method according to one embodiment compared to DAPI in a state-of-the-art tissue staining method. [Figure 17] Figure 17 shows the effect of multiple immunostaining on bulk staining images of whole mouse brain in the absence (iDISCO) and presence (INSIGHT) of the chaotropic ion [B12H12]2-, despite the use of identical tissue processing conditions. Detailed Description of the Invention

[0020] Embodiments of the present invention relate to an in situ supramolecular chemical system (INSIGHT) for improving penetration depth and uniformity in two-dimensional or three-dimensional immunostaining for the analysis of one or more target antigens in biological tissue, which may have any thickness. The system enables uniform deep immunostaining of thick tissue with optical tissue clearing, improving three-dimensional imaging using optical tomography techniques. The system includes a chaotropic salt for providing chaotropic ions, a probe, and optionally a complexing agent, and is used to perform three-dimensional immunohistochemistry.

[0021] The chaotropic salt may be perrhenate, closo-borane, closo-carborane, cobalt bis(dicarbollide) (CoSAN), or a polyoxometalate of tungsten, molybdenum, tantalum, niobium, or vanadium (e.g., phosphotungstate and phosphomolybdate). In one embodiment, the chaotropic salt is sodium dodecahydrododecaborate (NaB 12 H 12 ), but is not limited thereto. The salt may be, for example, a chaotropic anion [B 12 H 12 ] 2- or an anion derived from an unsubstituted closo-borane or closo-carborane having more than nine atoms in the cage structure, where the boron or carbon atoms in the cage may be independently substituted with fluoro, chloro, bromo, iodo, hydroxy, amino, or alkyl groups. The salt may have a cation, which may be, but is not limited to, any alkali metal or alkaline earth metal.

[0022] Chaotropic anions are used to inhibit aggregation with denatured antibodies and to inhibit the interaction of antigens with functional antibodies. Complexing agents that can be used with chaotropic anions include, but are not limited to, γ-cyclodextrin (γCD) (shown in Figure 2A), other cyclodextrins shown in Figure 2B, cucurbiturils, calixarenes, cyclophanes, cryptands, cryptophanes, their chemical derivatives, or oligomers with anion-associating repeating units.

[0023] The cyclodextrin may be hexakis-β-glucopyranose (also called α-cyclodextrin), heptakis-β-glucopyranose (also called β-cyclodextrin), octakis-β-glucopyranose (also called γ-cyclodextrin), or derivatives thereof, which have the general structure shown in FIG. 2B, in which the hydroxy groups in the structure may be substituted with amino, methoxy, (2-hydroxy)propoxy, sulfo, guanidino, acetato, acetamido, azide, bromo, iodo, chloro, toluenesulfonyl, thiol, succinic acid, phosphate, 4-sulfatobutoxy, carboxymethoxy, or (2-aminoethyl)amino groups.

[0024] The chaotropic salt is provided for co-incubation with a probe, which may be, but is not limited to, a primary antibody or antigen, as well as a functionalized antibody, such as a fluorescently labeled fragment antigen-binding secondary antibody (Fab) or single domain antibody (also called a VHH antibody or nanobody), which is selected for binding to a target primary antigen in the tissue sample. Probes can also be other proteins or small molecules, such as multiple fluorescently or dye-labeled lectins, dye-labeled streptavidin or avidin derivatives, dye-labeled protein tags (e.g., SNAP tags, CLIP tags, Spy tags), or dye-labeled protein binders designed by computer software or deep neural networks, cationic oligopeptides, DAPI (4',6-diamidino-2-phenylindole), Hoechst dyes, thiazole orange and its derivatives, oxazole blue and its derivatives, propidium iodide, eosin, lipophilic dyes (e.g., DiI, CM-DiI, DiD, DiR, and DiO), nuclear red, methylene blue, methyl green, cresyl violet, bromophenol red, alizarin red S, etc. S), and malachite green.

[0025] Chaotropic anions facilitate diffusion across cell membranes and inhibit binding of secondary antibodies to their own tissue antigen targets as they penetrate the tissue, allowing the probe secondary antibodies to diffuse freely throughout the tissue without being consumed by immobilized tissue antigens. The molecular host is [B 12 H 12 ] 2-The chaotropic anions and complexing agents may be provided in separate vehicles to anchor the chaotropic anions and associated antibodies within the tissue. The incubated tissue can then be transferred to a solution containing a molecular host complexing agent, which can then release the associated antibodies from their target antigen binding inhibition.

[0026] One embodiment of the present invention relates to a method for three-dimensional analysis of target antigens enabled by the INSIGHT system, and as shown in FIG. 3, chaotropic anions ([B 12 H 12 ] 2- ) are fluorescently labeled secondary antibodies Fab and [B 12 H 12 ] 2- The tissue is treated with the first staining buffer containing the primary antibody at room temperature for a desired time, resulting in the tissue being permeated with the antibody. The antibody-permeated tissue is then transferred to the second staining buffer containing a molecular host (shown as γ-CD in Figure 3), which diffuses into the tissue and forms [B 12 H 12 ] 2- Complexation with chaotropic anions releases antibodies bound to the Fab antibody, facilitating binding of the Fab antibody to its target antigen. Fluorescence imaging of labeled secondary antibodies with Fab-antigen pairs enables two-dimensional or three-dimensional imaging. Deep three-dimensional analysis is possible using tissue clearing agents and techniques, including but not limited to CLARITY, CUBIC, SWITH, SHIELD, BABB, iDISCO, pathoDISCO, uDISCO, vDISCO, 3DISCO, Fluoro-BABB, Ce3D, ScaleS, SeeDB, SeeDB2S, OPTIClear, OPTIClear2, SHANEL, or PEGASOS.

[0027] Supramolecular histochemistry methods according to embodiments address the limitations of conventional histochemical staining techniques, which often suffer from suboptimal signal-to-background and signal-to-nonspecific binding signal ratios due to uneven penetration and nonspecific binding of probes. Supramolecular histochemistry methods according to embodiments use a unique combination of reagents and processing steps to enable uniform and deep penetration of antibodies, lectins, and small molecule probes into specimens, thereby improving staining specificity and enhancing visualization of target structures.

[0028] Supramolecular histochemistry ensures uniform distribution of probes throughout the specimen, resulting in uniform penetration and thus consistent and precise staining of target structures, which is particularly important for thick or dense specimens where achieving sufficient penetration can be difficult with conventional staining methods.

[0029] High penetration uniformity results in unbiased quantitative data, which provides unbiased data across the entire penetration depth, thereby enabling a quantitative reflection of the ground truth signal for each detected biomolecular target. This is particularly important for thick or dense specimens, where achieving sufficient penetration with conventional staining methods can be difficult. High penetration uniformity results in unbiased quantitative data, which provides unbiased data across the entire penetration depth, thereby enabling a quantitative reflection of the ground truth signal for each detected biomolecular target.

[0030] The deep penetration of this method allows the probe to reach deep within the specimen, ensuring effective staining of structures located far from the surface. This is important for analyzing complex biological samples such as tissues and organs, where important information may be hidden deep within the specimen. By increasing staining specificity, supramolecular histochemistry can alter the nuances of antibody-antigen interaction dynamics, thereby simultaneously improving both the signal-to-background and signal-to-nonspecific binding ratios in immunofluorescence.

[0031] Supramolecular histochemistry provides a clear and unambiguous visualization of target structures, which is particularly important when detecting low-abundance targets, using difficult-to-react antibodies, or when distinguishing structures that are similar to each other or distinguishing signal from autofluorescence.

[0032] Supramolecular histochemistry has near-universal applicability to probes and tissues, is compatible with a wide range of commercially available probes, including antibodies, lectins, and small molecule probes, and is applicable to a wide variety of tissues, from plants to animals, and even clinically derived archival and fresh-frozen tissues, organoids, chimeric tissues, excised tissues, and xenografts, making it a versatile method for research and clinical applications. In situ supramolecular chemistry offers a simple and easy-to-operate chemical tool for controlling the behavior of probes throughout tissues in complex environments. It is also extremely simple to implement, requiring no specialized equipment or specialized knowledge; it can be performed by simply adding a few well-defined, low-cost additives to standard staining solutions. This is crucial as automation, scalability, reliability, cost, parallel processing, and market viability are key factors for future tissue diagnostic applications.

[0033] Three-dimensional histological observation of tissues can reveal previously unknown principles of structural organization, potentially contributing to improved diagnosis and prognosis of disease during biopsy and resection. However, despite advances in whole-animal and human organ clearing technologies, the depth to which probes can penetrate tissue is typically limited to a few hundred micrometers. Antibodies, in particular, exhibit significant variability in performance across different conditions and concentrations. This poses the greatest obstacle to the expansion of 3D histology, limiting its application to sequential sectioning and staining or to transgenic animals with endogenous fluorescence when using tissue clearing.

[0034] While recent methods address this challenge, they lack key characteristics required for an ideal method, including staining quality, reliability, scalability, ease of use, speed, compatibility with multiplexed and multimodal labeling, and cost. These shortcomings hinder widespread adoption in research and clinical settings. Furthermore, most methods lack sufficient signal uniformity across the entire penetration depth, complicating quantitative assessment of protein expression levels. In summary, despite the potential utility of 3D histological methods, the probe penetration depth currently limits their application. Existing penetration-improvement methods lack key characteristics essential for widespread adoption, highlighting the need for novel solutions.

[0035] The supramolecular histochemistry method described herein overcomes these technical bottlenecks, enabling a user-friendly 3D histochemistry method with the following features: uniform probe penetration up to centimeters deep, quantitative and highly specific immunostaining signals, rapidity, low cost, and high scalability, adaptable to tissue sizes and shapes, and simple immersion-based staining, making it easily adoptable in any laboratory and suitable for automation. Furthermore, it can use commercially available antibodies or probes and is directly applicable to wild-type mouse and human tissues. This suite of techniques utilizes an in situ supramolecular reaction system, including the boron cluster compound closo-dodecahydrododecaborate and a γ-cyclodextrin derivative, to reversibly modulate probe-target interactions throughout the tissue, achieving uniform and deep-penetrating 3D histochemistry.

[0036] To design a reliable, automatable, and scalable 3D histochemistry method, chemical approaches were employed to enhance molecular mobility within the tissue matrix. The use of reversible, bioorthogonal, noncovalent click chemistry in supramolecular systems is ideally suited for this technique. Based on a reaction-diffusion model of probe transport within tissues, two complementary yet compatible supramolecular histochemistry approaches have been proposed: a switchable chaotropic system for global control of intermolecular interactions, and a supramolecular carrier approach for creating flow channels for the selective transport of specific probes throughout the tissue matrix.

[0037] In 3D immunostaining, the relative rates of various antibody-related chemical reactions (e.g., antigen binding, nonspecific tissue binding, aggregation) and their diffusive kinetics determine the ultimate distribution of antigen-bound antibodies within the tissue matrix. In the switchable chaotropic approach, chaotropes, known to reduce solvent viscosity and overall protein-protein interactions, facilitate probe diffusion within the matrix. After the protein probe is uniformly distributed throughout the tissue, the chaotrope can be bioorthogonally removed by an enthalpy-driven complexation process using a supramolecular host with a compatible pore size. The restoration of the ordered water structure re-provides the driving force for antibody-antigen interactions throughout the tissue. In the supramolecular carrier approach, a mobile supramolecular host complexes the probe as a guest molecule, facilitating its partitioning into the solution phase rather than a fixed target within the tissue.

[0038] This corresponds to an additional step, host-guest dissociation, which slows the reaction rate between the probe and its target in the tissue, thereby facilitating probe diffusion. The molecular host can also be viewed as a molecular-sized "organic solvent pocket" that facilitates dissolution of the probe-target precipitate. This effect, compared to organic solvents, has minimal impact on protein function, which is essential for immunostaining.

[0039] For application in the switchable chaotropic approach, highly chaotropic and weakly coordinating anions were screened to provide sufficient enthalpic driving force for in situ supramolecular reactions while minimizing protein denaturation through enthalpy-driven structural melting. Using standard deep immunostaining benchmarking protocols, several supramolecular systems, namely perrhenate / α-cyclodextrin (ReO4 - / αCD), closo-dodecaborate ion [B 12 X 12 ] 2- / γCD (where X is H, Cl, Br, or I), metallacarborane [Co(7,8-C2B9H 11 )2] - / γCD, polyoxometalate [PM 12 O 40 ] 3- The suitability of ReO4 / γCD (where M is Mo or W) (shown in Figure 1B) and deoxycholate / βCD for in situ deep immunohistochemistry was compared. - , [B 12 H 12 ] 2- , [Co(7,8-C2B9H 11 )2] - Only deoxycholate and deoxycholate were demonstrated to be compatible with immunostaining conditions without causing tissue damage or precipitation, whereas the deoxycholate / βCD system resulted in undesirable intravascular precipitation.

[0040] ReO4 - , [B 12 H 12 ] 2- , and [Co(7,8-C2B9H 11 )2] - When comparing the performance of [B 12 H 12 ] 2- The best results in terms of staining sensitivity, specificity, and signal uniformity across the depth direction were obtained when using / γCD, and the effect of derivatization with γCD was negligible. [B 12 H 12 ] 2- The simple substance and the corresponding supramolecular complex [B 12 H 12 ] 2- The differential modulation of antibody-antigen interactions by ⊃2HP-γCD was verified using a SARS-CoV-2 rapid antigen test as a reaction-diffusion implementation model (see Figure 13). A deep immunostaining step in the INSIGHT protocol was established using the more water-soluble 2-hydroxypropylated derivative (2HP-γCD) (see FIG. 14). In this step, first select [B12 H 12 ] 2- The procedure involves incubating tissue with antibodies in 2HP-γCD / PBS, followed by incubation in 2HP-γCD / PBS. The entire process involves a simple exchange of two buffer solutions using low-hazard chemicals, and can be performed at incubation temperatures ranging from 20 to 37°C without the need for special equipment.

[0041] For small molecule dyes, size-matched and charge-complementary cyclodextrin derivatives are cost-effective supramolecular host carriers effective for deep tissue penetration. For example, as shown in Figure 15, sulfobutyl-etherified βCD (SBE-βCD) can form high-affinity supramolecular complexes with predominantly positively charged nucleic acid probes. The formed mobile [probe ⊂ SBE-βCD] species exhibited enhanced penetration in INSIGHT (Figure 16), and [probe ⊂ SBE-βCD] species exhibited enhanced penetration in INSIGHT (Figure 17). n+ ] / [B 12 H 12 2― ] to inhibit the formation of precipitates. Furthermore, the mobility of the [probe ⊂ xCD] complex was enhanced in the protein matrix, boiled egg white, confirming that nonspecific interactions between the probe and tissue components are an important factor in the reaction-diffusion model. Therefore, by replacing non-denaturing detergents (e.g., Triton X-100 and Tween-20) with boron cluster-based host-guest systems and substituting small molecule probes with probe ⊂ host complexes, it is possible to extend traditional section-based histochemistry to three dimensions.

[0042] The INSIGHT System improves immunostaining quality compared to prior art systems by maximizing specific staining, minimizing nonspecific staining, improving overall signal intensity, increasing immunostaining penetration depth in thick tissues, improving immunostaining uniformity throughout the penetration depth in thick tissues, and improving protein signal intensity (typically, but not necessarily, fluorescent in tissues). Maximizing specific staining refers to obtaining a higher signal in areas where the antibody is expected to produce a signal. This can be based on theoretical predictions based on published immunostaining examples, antibody vendor information, existing databases, or expert knowledge of the secondary antibody and its antigen under the specific experimental conditions being used.

[0043] Minimizing nonspecific staining refers to reducing or eliminating signal in areas where signal generation is not expected. Improving overall signal intensity refers to increasing the sum of signals obtained from the entire sample. Increasing immunostaining penetration depth refers to the ability to detect specific staining at greater distances from the tissue surface compared to current histological systems. Improving immunostaining uniformity across penetration depth means that the signal intensity and contrast of specific staining in deeper tissues is superior to that in shallower tissues, i.e., the deep-to-shallow signal ratio is closer to 1 than with other systems. Improving fluorescent protein signal intensity in tissues applies to tissues that endogenously express fluorescent proteins. The increased signal and image contrast can be either endogenous or exogenous.

[0044] Quantitative benchmarking of INSIGHT's performance against other experimental techniques was performed using a rigorous experimental design. An overview is shown in Figure 4, and the results are presented in Figure 5. This allows us to derive optimal conditions for various supramolecular hosts and chaotropic anion compositions for INSIGHT by comparing the fluorescence intensity of planar images obtained by bulk staining of the entire tissue with that of cross-section images obtained by cutting the specimen at the corresponding position in the image plane and then staining it. Analyzing the change in fluorescence intensity with depth allows us to derive optimal conditions for INSIGHT regarding various supramolecular hosts and chaotropic anion compositions. This process is illustrated in Figure 6. This benchmarking experiment (Figure 4) was conducted in two stages. In the first stage, adult mouse brains were stained with deep immunostaining (bulk staining) to form cross-sections. In the second stage, the formed cross-sections were restained with primary antibodies in standard immunostaining buffer (1x PBS) containing 0.1% Tween-20 (this process is called cut-staining). INSIGHT was found to achieve the deepest immunostaining and the best uniformity throughout the penetration depth, resulting in near-ideal staining, as shown in Figure 7. This is superior to current deep imaging methods, as shown in Figure 8. A comparison of cut staining signal intensity and cut staining penetration distance was performed, as shown in Figures 9 and 10.

[0045] Unlike other techniques such as SHANEL and CUBIC-HV, INSIGHT does not rely on tissue permeabilization.

[0046] INSIGHT's function is to reduce the motion drag of antibodies by lowering the viscosity within the tissue, thereby suppressing their consumption by immobilized tissue antigens during the tissue distribution phase, eliminating the reaction barrier and enhancing the diffusivity of Fab antibodies for deep and uniform transport throughout the tissue. The chaotropic effect, through specific complexation with γCD, results in an enthalpy gain in bulk water, which "deactivates" the chaotropic anions and releases their inhibition of antibody-antigen binding during the subsequent staining step, thereby acting as a rapid chemical "switch."

[0047] The versatility of INSIGHT was excellent compared to the other systems tested. INSIGHT was particularly outstanding, as shown in Figure 11, with compatibility confirmed for 85 of the 90 antibodies tested, the most compatible. Furthermore, for 72 of the 90 antibodies tested, INSIGHT outperformed any conventional immunostaining method. This is due to the fact that the INSIGHT method [B 12 H 12 ] 2- This is consistent with the molecular "chaperone" effect of the -γCD system, which refolds and restores the functionality of commercially available primary antibodies denatured by chaotropic anions. INSIGHT is particularly advantageous in that it is easy to operate (see Figure 12), requires short tissue processing times, is inexpensive, and does not require specialized equipment, making it fully automatable and easily accessible to any basic research laboratory.

[0048] INSIGHT complements conventional clearing reagents, which require deep immunostaining to demonstrate their clearing performance. INSIGHT addresses the bottleneck in implementing 3D immunofluorescence analysis: achieving sufficient penetration of antibody probes at reasonable cost and labor efficiency. Clinical application will be facilitated by an automated 3D imaging system that combines efficient tissue clearing, deep immunostaining, and high-speed optical tomography imaging.

[0049] Current state-of-the-art, automatable tissue clearing and optical tomography imaging technologies can be enhanced by INSIGHT by: providing reliable immunofluorescence signals with high specificity and low nonspecific signal; achieving reproducible penetration depth with uniform staining across the greatest possible depth; low cost; processing speeds of less than 48 hours; scalability across specimen sizes; and ease of operation that allows for automation.

[0050] As shown in Table 1 below, of all currently available deep staining technologies, only INSIGHT meets all of the criteria required for clinical use described above.

[0051] Table 1. Comparison of the practicality of various deep immunostaining systems for application to clinical specimens and tissue diagnosis in 3D histological analysis. JPEG2026502043000002.jpg68153

[0052] This novel technology has extremely broad applicability and is expected to make significant contributions to the fields of tissue diagnosis and research. In diagnostics, uniform penetration throughout the tissue specimen enables the quantification of dozens of markers in a highly accurate and standardized manner, reducing false positives and false negatives and enabling more definitive disease diagnoses from biopsy samples. Deep penetration provides information on early-stage lesions and intra-tissue heterogeneity that are often overlooked by conventional superficial analysis. Combined with the technology's simple workflow, cost-effectiveness, and potential for high-throughput applications, it promises to revolutionize tissue-based disease diagnosis.

[0053] In research, unbiased, quantitative 3D mapping based on multiple markers enables visualization of tissue physiological structure with unprecedented resolution, providing new insights into how disease alters tissue architecture in situ. This combination of high-throughput, parallelizable workflow, deep penetration, and quantitative analysis capabilities will enable new advances in drug discovery and personalized medicine. For example, it will aid in the discovery of potential biomarkers that suggest new indications for existing drugs. Furthermore, this technology provides a means to directly measure drug responses in situ throughout 3D tissue models and organoids, enabling the design of more effective and targeted therapeutics. Its simple workflow, cost-effectiveness, and applicability to large sample processing open new avenues for clinical diagnostics and large-scale screening, potentially contributing to earlier disease detection and improved patient outcomes.

[0054] This supramolecular histochemistry method has the potential to revolutionize the field of histochemistry by providing more effective and reliable staining techniques for analyzing biological specimens. Future developments may see the emergence of disruptive chemical approaches that make 3D histology more accessible and widespread in mainstream research and tissue diagnostics. This technology will enable researchers and clinicians to obtain high-resolution 3D images, potentially leading to a deeper understanding of disease. Furthermore, the simplicity and automation of this process will enable even researchers with minimal training to perform tissue analysis. As a result, this technology has the potential to democratize 3D histology, opening new research frontiers, fostering collaboration and knowledge sharing, and ultimately contributing to improved diagnostics, therapeutic advances, and patient outcomes through personalized medicine.

[0055] Supramolecular histochemistry can be performed using two different strategies: chaotropic or complexation. In the chaotropic strategy, primary antibodies from different host animals, such as rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, llama, camel, and horse, can be mixed and added directly to the staining solution during multiplex indirect immunohistochemistry. These can also be combined with monovalent secondary antibody Fab fragments from rat, mouse, pig, goat, rabbit, sheep, guinea pig, donkey, llama, camel, horse, and cow, or single-domain antibodies (nanobodies) from llama or alpaca, corresponding to the respective primary antibodies.

[0056] It is also suitable for signal amplification in combination with immunohistochemistry. For example, a biotin-labeled secondary antibody reagent can be used, followed by the addition of fluorescently labeled streptavidin in any subsequent step. This can be used in combination with chaotropes (supramolecular complex-forming host molecules), staining reagents, lectins, or other antibodies to enhance signal intensity. However, it should not be used in direct combination with a biotin-labeled secondary antibody reagent. This strategy is also compatible with lectin histochemistry, where the penetration depth, uniformity, and staining quality of one or more fluorescently or dye-labeled lectins can be improved by supramolecular histochemistry.

[0057] Supramolecular histochemistry can be performed in repeated cycles on the same tissue section, allowing multiple different images to be obtained from the same organ by using chaotropes in combination with sodium sulfite or β-mercaptoethanol to elute antibodies from the tissue block and allow a new round of supramolecular histochemistry to be performed.

[0058] This method is applicable to tissues that express endogenously fluorescent proteins without degrading their fluorescent signals, and can be used in conjunction with existing histological staining, immunohistochemistry, and lectin histochemistry techniques to enhance their performance.

[0059] After supramolecular histochemistry, tissue remains intact and its structure is not disrupted, making it suitable for downstream analytical steps, such as nucleic acid extraction and analysis by PCR, Northern and Southern blotting, in situ hybridization (ISH), RNA or DNA sequencing, or protein extraction and analysis by Western blotting, gel electrophoresis, mass spectrometry, and ELISA. Processed tissues can then be physically sectioned and imaged, or optically cleared and viewed.

[0060] Supramolecular histochemistry is compatible with, and can be performed before, during, or after, the following processes: Tissue immunohistochemistry processing, immunocytochemistry processing, in situ hybridization processing, fluorescent in situ hybridization processing, chromosome identification processing, staining processing, antigen retrieval processing, blocking processing, cytochemistry processing, molecular chemistry processing, epitope retrieval processing, preprocessing step, tissue permeabilization processing, tissue clearing processing, tissue dehydration processing, tissue rehydration processing, tissue delipidation processing, tissue fixation processing, tissue washing processing, and / or tissue recovery processing from paraffin blocks.

[0061] This method allows for the establishment of an automatable, rapid, scalable, and cost-effective protocol for obtaining multiply stained whole tissue blocks for 3D tissue diagnosis in research and clinical settings.

[0062] Methods and Materials

[0063] [General supramolecular histochemistry protocol] Any fixed biological tissue specimen can be used. Appropriate tissue pretreatment can be performed as needed. Once thoroughly washed, tissue can be directly subjected to supramolecular histochemistry, similar to conventional histochemistry.

[0064] The tissue is pre-incubated in a solution of the desired composition selected by the user, with 0.01-1 M chaotrope, at 15-55°C for 2 minutes to 12 hours, with shaking as needed. Detection of biomolecular targets is performed using antibodies, lectins, or low-molecular-weight dyes, which are optionally pre-complexed with a supramolecular host, in the presence of 0.01-1 M chaotrope, in addition to the standard staining solution composition, and incubated at 10-37°C for 5 minutes to 2 weeks, with shaking as needed.

[0065] The staining solution is then diluted with a solution of the desired composition or replaced with a wash solution and incubated at 4-37°C for 5 minutes to 1 week, which can contain a supramolecular host molecule that complexes with any remaining chaotropes in the tissue.

[0066] Subsequent imaging, histological analysis, or tissue analysis can be performed as usual, but supramolecular histochemistry has the advantage that multiplex immunohistochemical signals, small molecule probe signals, and lectin histochemical signals are more uniformly distributed and penetrate deeper, increasing the specificity of staining and improving the signal-to-background ratio relative to nonspecific background signals.

[0067] This process can be repeated by combining it with a treatment to recover the stained tissue. For example, if optical tissue clearing has been performed, the tissue can be recovered by washing with a standard solution to remove the clearing, and then eluting the antibody using a solution containing a chaotrope and a reducing agent (e.g., sulfite ions, β-mercaptoethanol, or tris(2-carboxyethyl)phosphine). These treatments can be carried out in any solution with the desired composition at 20–55°C for 1 hour to 1 week. After washing, another round of supramolecular histochemistry can be performed again as described above.

[0068] Mouse whole brains were perfused with 4% paraformaldehyde and post-fixed overnight at 4°C. They were then washed three times at room temperature for 1 hour each with phosphate-buffered saline (PBSN) containing 0.02% (w / v) sodium azide. As a pretreatment, mouse whole brains were dehydrated through graded methanol, defatted overnight in a dichloromethane:methanol (2:1, v / v) mixture, and then rehydrated again through graded methanol.

[0069] The tissue was then transferred to PBSN, immersed in PBSN containing 0.2 M sodium closo-dodecahydrododecaborate, and incubated overnight at 37°C with gentle shaking. The solution was then replaced with a new solution containing 0.2 M sodium closo-dodecahydrododecaborate in PBSN, to which 10 μg of a rabbit-derived primary antibody targeting an antigen in the tissue and 10 μg of a donkey anti-rabbit Fab fragment (secondary antibody) labeled with a fluorescent dye (e.g., Alexa Fluor 647) were added.

[0070] Based on the volume of primary and secondary antibody reagents added, the concentration of the chaotrope closo-dodecahydrododecaborate can be adjusted to the desired level by supplementing the appropriate amount of a 10x solution containing 2.5 M sodium closo-dodecahydrododecaborate in PBSN.

[0071] The tissues were then incubated at room temperature for 3 days, transferred to a solution containing 0.25 M 2-hydroxypropyl-γ-cyclodextrin for 1 day, washed in PBSN for at least 1 hour, dehydrated through graded methanol, and cleared with benzyl alcohol / benzyl benzoate (BABB). Finally, staining of the target antigens was imaged using a selective plane illumination microscope.

[0072] 1×1×1cm 3 Human tissue specimens were obtained from surgical specimens and immersion-fixed in 10% neutral-buffered formalin. The tissues were washed in PBS, dehydrated through graded ethanol series, treated overnight in dichloromethane:methanol (2:1, v / v), and then rehydrated through graded ethanol series. The tissues were then washed in PBS and placed in 1x TBE buffer containing 0.1M potassium closo-dodecahydrododecaborate, 0.1% (w / v) eosin Y, and 0.1M triethanolamine or 0.1M N-methylglucamine for overnight staining.

[0073] The tissues were then transferred to 0.25 M 2-hydroxypropyl-γ-cyclodextrin solution containing 50% (v / v) tetrahydrofuran and 0.1 M N-methylglucamine in 1x TBE buffer and incubated overnight. The tissues were then transferred to 100% tetrahydrofuran for dehydration and cleared with dibenzyl ether. Finally, the tissues were imaged using a confocal microscope to observe general tissue morphology.

[0074] [Single antibodies and small molecule dyes] Whole mouse kidneys were removed and fixed in graded methanol, then treated with dichloromethane, rehydrated again in graded methanol, and post-fixed in 4% paraformaldehyde at 4°C for 2 hours.

[0075] The kidneys were washed for 1 hour in phosphate-buffered saline (PBS) containing 0.02% (w / v) sodium azide in PBSN, then immersed in PBSN containing 0.2 M lithium closo-dodecahydrododecaborate and incubated overnight at 37°C with gentle shaking.

[0076] The incubation solution was replaced with fresh PBSN containing 0.25 M sodium closo-dodecahydrododecaborate and 1 μg of mouse-derived primary antibody targeting an antigen in the tissue, and 1 μg of Fab fragment of donkey anti-mouse secondary antibody labeled with a fluorescent dye (e.g., CF488A).

[0077] At the same time, an aqueous solution of DAPI dilactate salt was mixed with sulfobutyl etherified β-cyclodextrin in a separate tube for 10 seconds to form a complex, which was then quickly added to the antibody staining solution.

[0078] Based on the volume of primary antibody, secondary antibody, and nucleic acid stain added, an appropriate amount of PBSN containing 2.5 M sodium closo-dodecahydrododecaborate can be supplemented to adjust the concentration of the chaotrope closo-dodecahydrododecaborate to the desired level.

[0079] The tissues were then incubated overnight at room temperature, transferred to a solution containing 0.2 M 2-hydroxypropyl-γ-cyclodextrin for 3 hours, washed with PBSN for at least 1 hour, dehydrated in graded methanol, and cleared in benzyl alcohol / benzyl benzoate (BABB) clearing solution. The cleared kidneys were imaged based on antigen staining using a selective plane illumination microscope.

[0080] [Use of a single antibody, multiple lectins, and small molecule dyes] Whole mouse kidneys were perfusion-fixed with 4% paraformaldehyde in PBS, permeabilized with 10 mM sodium deoxycholate in 0.1 M Tris buffer, and thoroughly washed with 0.1 M Tris buffer. The tissues were then transferred to a solution containing 0.25 M cesium closo-dodecahydrododecaborate in 0.1 M HEPES buffer, pH 8, and incubated at 37°C for 1 hour with gentle shaking.

[0081] The incubation solution was replaced with a new solution containing 0.25 M cesium closo-dodecahydrododecaborate in 0.1 M HEPES buffer, pH 8, to which 1 μg of a guinea pig primary antibody targeting a tissue antigen and 1 μg of a goat anti-guinea pig secondary antibody Fab fragment labeled with Atto 490LS fluorescent dye were added. Simultaneously, an aqueous solution of oxazole blue was complexed with sulfobutyletherified β-cyclodextrin in a separate tube for 10 seconds and then immediately added to the antibody staining solution.

[0082] Simultaneously, 10 μg of Griffonia simplicifolia lectin I labeled with AlexaFluor 488 dye, 5 μg of Phaseolus vulgaris agglutinin lectin labeled with Atto 647N dye, and 10 μg of succinated wheat germ agglutinin (WGA) labeled with tetramethylrhodamine were added to the staining solution containing the antibody and supramolecularly complexed nucleic acid probe.

[0083] Based on the volume of primary antibody, secondary antibody, and nucleic acid stain added, an appropriate amount of PBSN containing 2.5 M sodium closo-dodecahydrododecaborate can be added to adjust the concentration of closo-dodecahydrododecaborate to the desired level.

[0084] The tissues were then incubated at room temperature for 6 hours, transferred to a solution containing 0.25 M 2-hydroxypropyl-γ-cyclodextrin for 3 hours, and washed extensively with PBSN. Finally, the tissues were cleared with OPTIClear solution and imaged by confocal microscopy.

[0085] [Use of multiple antibodies, multiple lectins, and multiple small molecule dyes] Two-mm-thick mouse brain tissue sections were obtained from the archive and dehydrated using 4% sodium dodecyl sulfate or dichloromethane. The tissues were washed with PBSN, immersed in 0.25 M sodium closo-dodecahydrododecaborate, and incubated at 32°C for 2 hours.

[0086] The incubation solution was then replaced with fresh 0.25 M sodium closo-dodecahydrododecaborate and staining was carried out, to which the following primary probes were added: Rabbit primary antibody 3 μg 3 μg of mouse primary antibody of the IgG1 isoform 1 μg of mouse primary antibody of the IgG2a isoform Guinea pig primary antibody 3 μg ·Goat-derived primary antibody 5 μg Chicken-derived primary antibody 2 μg Rat primary antibody 2 μg 3 μg of tomato (Lycopersicon esculentum) lectin labeled with AlexaFluor 700 dye 3 μg of peanut agglutinin labeled with 7-aminocoumarin dye 1 pmole of Hoechst 33342 dye complexed with 2.5 pmoles of sulfobutyletherified β-cyclodextrin

[0087] Based on the volume of dye probe reagent added, an appropriate amount of 2.5M sodium closo-dodecahydrododecaborate in PBSN was added to adjust the concentration of closo-dodecahydrododecaborate to the desired level.

[0088] Additionally, the following secondary antibody Fab fragments were added to the staining mixture: 3 μg of donkey anti-rabbit secondary antibody Fab fragment conjugated with Atto 430LS dye 3 μg of goat anti-mouse IgG1 secondary antibody Fab fragment conjugated with AlexaFluor 555 dye 1 μg of goat anti-mouse IgG2a secondary antibody Fab fragment conjugated with AlexaFluor 594 dye 3 μg of donkey anti-guinea pig secondary antibody Fab fragment labeled with Atto 490LS dye 5 μg of bovine anti-goat Fc-specific secondary antibody Fab fragment conjugated with BODIPY TMR dye 2 μg of donkey anti-chicken IgY secondary antibody Fab fragment conjugated with AlexaFluor 647 dye 2 μg of donkey anti-rat secondary antibody Fab fragment conjugated with AlexaFluor 680 dye

[0089] After staining, the tissues were incubated for 1 hour, then washed with 0.25 M 2-hydroxypropyl-γ-cyclodextrin in PBSN, followed by another wash in PBSN, dehydration, and optical clearing in a benzyl alcohol / benzyl benzoate mixture. The cleared tissues were imaged using a hyperspectral confocal imaging platform.

[0090] [Use of multiple antibodies and multiple lectins without supramolecular complexation] Fixed, 2-mm-thick mouse brain tissue sections obtained from the archive were used. The tissues were dehydrated, defatted with 4% sodium dodecyl sulfate solution or dichloromethane, washed with PBSN, and then immersed in 0.25 M sodium closo-dodecahydrododecaborate solution and incubated at 37°C for 2 hours.

[0091] The incubation solution was renewed with 0.25 M sodium closo-dodecahydrododecaborate solution, and staining was performed.

[0092] The staining solution contained the following: Rabbit primary antibody 3 μg 3 μg of tomato (Lycopersicon esculentum) lectin labeled with AlexaFluor 700 dye 3 μg of peanut agglutinin labeled with 7-aminocoumarin dye 1 pmole of Hoechst 33342 dye complexed with 2.5 pmoles of sulfobutyletherified β-cyclodextrin

[0093] Based on the volume of staining probe reagent added, an appropriate volume of 2.5 M sodium closo-dodecahydrododecaborate in PBSN was added to adjust the concentration of closo-dodecahydrododecaborate to the desired level. Additionally, 3 μg of donkey anti-rabbit secondary antibody Fab fragment labeled with Atto 430LS dye was added to the staining mixture. After staining, the tissue was incubated for 6 hours.

[0094] The sections were then washed with PBSN to neutralize the effect of closo-dodecahydrododecaborate, followed by another wash with PBSN. After dehydration, the sections were cleared with a benzyl alcohol / benzyl benzoate mixture and imaged using a confocal imaging platform.

[0095] [Use of multiple antibodies, multiple lectins, and multiple small molecule dyes] Tissues were treated with any combination of staining reagents as described above, followed by optical tissue clearing and imaging. The tissues were then washed in methanol, rehydrated through graded methanol, and thoroughly washed with PBS. They were then subjected to another cycle of supramolecular histochemistry using any combination of staining reagents as described above. They were then again treated with optical clearing and imaging. This process was repeated eight times on the same tissue specimen.

[0096] [Use of multiple antibodies, lectins, and small molecule dyes followed by downstream applications (FISH)] Tissues were processed with any combination of staining reagents, optically cleared, and imaged as described above. They were then analyzed by single-molecule fluorescence in situ hybridization (smFISH) for the detection of transcript molecules within the tissue. smFISH was performed using either the hairpin chain reaction (HCR) method as previously described or commercially available systems such as Stellaris or ACDLabs RNAScope.

[0097] A 3.5 cm x 2.5 cm x 1.8 cm piece of human medulla oblongata tissue was removed from a cadaver and fixed in neutral buffered formalin for two weeks, then thoroughly washed with PBS. To record histological images, the tissue was embedded in 2% agarose and imaged using magnetic resonance imaging (MRI). After imaging, the agarose gel was manually removed to recover the sample, which was then thoroughly washed with PBSN. The tissue was then incubated in PBSN containing 0.5 M sodium closo-dodecahydrododecaborate at 37°C for one week with gentle shaking. The solution was then replaced with PBSN containing 0.25 M sodium closo-dodecahydrododecaborate, and the following reagents were mixed and incubated at room temperature for one week with gentle shaking: Rabbit primary antibody (100 μg) Mouse primary antibody (50 μg) Tetramethylrhodamine-labeled Griffonia simplicifolia I lectin (100 μg) DAPI dilactate (50 pmoles) complexed with sulfobutyl etherified β-cyclodextrin 0.1% (w / v) Eosin Y solution complexed with γ-cyclodextrin AND ONE OF THE FOLLOWING: AlexaFluor 488-conjugated donkey anti-rabbit secondary antibody Fab fragment (100 μg) SeTau 647-conjugated donkey anti-mouse secondary antibody Fab fragment (50 μg)

[0098] The tissues were then washed in a solution containing 0.25 M 2-hydroxypropyl-γ-cyclodextrin, 0.1 M γ-cyclodextrin, and PBSN at room temperature for 1 week with gentle shaking, and then washed again in PBSN for 2 days at room temperature. Finally, the tissues were imaged using two-photon tomography.

[0099] [Examples of FFPE-derived human tissue] Portions of breast cancer tissue were retrieved from paraffin-wax-embedded blocks that had previously been formalin-fixed. The blocks were heated to <100°C to dissolve the wax and release the tissue, followed by extensive washing with xylene or limonene to remove residual wax. The tissue was then degreased with dichloromethane / methanol, rehydrated through graded isopropanol, washed with water and PBSN, and then subjected to multiple supramolecular histochemistry, similar to the "Use of multiple antibodies, multiple lectins, and multiple small molecule dyes" experiment described above.

[0100] [Examples of fresh-frozen tissue] High-grade glioma tissue sections were freshly obtained from neurosurgery and stored at -80°C until transported to our facility for processing. The tissues were then thawed, fixed overnight in 4% paraformaldehyde in PBS, dehydrated through graded methanol, defatted in dichloromethane / methanol, rehydrated through graded methanol, and washed with PBSN. They were then subjected to multiple supramolecular histochemistry processing, similar to the "Use of multiple antibodies, multiple lectins, and multiple small molecule dyes" experiment described above.

[0101] All documents mentioned or cited in this specification, including drawings and tables, are incorporated herein by reference in their entirety, to the extent that they do not contradict the explicit statements herein. The examples and embodiments described herein are for illustrative purposes only, and it should be understood that various modifications and variations may occur to those skilled in the art based on these examples, and are intended to be included within the spirit and scope of this application. Furthermore, any invention or element or limitation of an embodiment thereof disclosed herein may be combined, individually or in any combination, with any other invention or element or limitation of an embodiment thereof disclosed herein, and all such combinations are considered within the scope of the present invention, without limiting its scope.

[0102] Example

[0103] [Example 1] A supramolecular histochemistry system for staining, comprising at least one probe, at least one chaotropic ion, and optionally at least one complexing agent, wherein the at least one probe achieves penetration throughout the tissue with uniform distribution within the tissue to achieve high specificity of tissue staining.

[0104] [Example 2] The supramolecular histochemistry system for staining described in Example 1, wherein the at least one probe is a plurality of probes, the at least one chaotropic ion is a single chaotropic ion, and / or, optionally, the at least one complexing agent is a plurality of complexing agents.

[0105] [Example 3] The supramolecular histochemistry system for staining described in Example 1, wherein the chaotropic ion is a derivative of unsubstituted closo-borane with more than 9 atoms, an unsubstituted closo-carborane with more than 9 atoms, any mixture thereof, or any derivative thereof in which the boron or carbon atoms are independently substituted with hydrogen, fluorine, chlorine, bromine, iodine, a hydroxy group, an amino group, or an alkyl group.

[0106] [Example 4] The supramolecular histochemistry system for staining as described in Example 3, wherein the chaotropic ion is closo-dodecaborate.

[0107] [Example 5] The supramolecular histochemistry system for staining described in Example 1, wherein the complexing agent comprises a cyclodextrin, a cucurbituril, a calixarene, a cyclophane, a cryptand, a cryptophane, a chemical derivative of any of these, an oligomer having an anion-associative repeating unit, or any mixture thereof.

[0108] [Example 6] The supramolecular histochemistry system for staining described in Example 5, wherein the cyclodextrin is hexakis-β-glucopyranose (α-cyclodextrin), heptakis-β-glucopyranose (β-cyclodextrin), or octakis-β-glucopyranose (γ-cyclodextrin), and optionally, one or more of the hydroxy groups are independently substituted with an amino group, a methoxy group, a (2-hydroxy)propoxy group, a sulfato group, a guanidino group, an acetato group, an acetamide group, an azido group, a bromo group, an iodo group, a chloro group, a toluenesulfonyl group, a thiol group, a succinyl group, a phosphato group, a 4-sulfatobutoxy group, a carboxymethoxy group, or a (2-aminoethyl)amino group.

[0109] [Example 7] The supramolecular histochemistry system for staining described in Example 6, wherein the cyclodextrin is (2-hydroxypropyl)-γ-cyclodextrin.

[0110] [Example 8] The supramolecular histochemistry system for staining described in Example 1, wherein the plurality of probes comprises a primary antibody, an antigen, a lectin, a functionalized antibody, an oligopeptide, a small molecule dye, and / or a fluorescent DNA stain.

[0111] [Example 9] The supramolecular histochemistry system for staining described in Example 1 further comprises a chemical buffer system.

[0112] [Example 10] The supramolecular histochemistry system for staining described in Example 9, wherein the chemical buffer system comprises Good's buffer, phosphate buffered saline (PBS), Tris, CAPS, HEPES, or any combination thereof.

[0113] [Example 11] The supramolecular histochemistry system for staining according to Example 10, wherein the chemical buffer system has a pH of 6 to 11 and a concentration of 10 to 1000 mM.

[0114] [Example 12] The supramolecular histochemistry system for staining described in Example 9, wherein the chemical buffer system comprises 1x PBS at pH 7-8.

[0115] [Example 13] Supramolecular histochemistry for staining as described in Example 1, where the chaotropic ion and the complexing agent are provided in separate vehicles.

[0116] [Example 14] providing a supramolecular histochemical system for staining as described in Example 1; providing a specimen comprising tissue; incubating a specimen comprising the tissue with chaotropic ions of the supramolecular histochemical system to form an incubated specimen; combining the incubated sample with a system comprising a chemical buffer system and, optionally, a complexing agent of the supramolecular histochemical system to promote association of the probe with the target to generate a signal; imaging said signal.

[0117] [Example 15] The method of example 14, wherein the combining step is dilution without a complexing agent.

[0118] [Example 16] The method according to Example 14, wherein the tissue-containing specimen is a tissue section having a thickness of about 1 to about 50,000 μm obtained by frozen sectioning, vibration sectioning, or paraffin sectioning, or is processed as an unprocessed organ.

[0119] [Example 17] 15. The method of Example 14, wherein the tissue-containing specimen is fixed by formalin fixation, glutaraldehyde fixation, methanol fixation, ethanol fixation, glyoxal fixation, picric acid fixation, trichloroacetic acid fixation, polyacrylamide-formalin fixation, polyglycerol 3-polyglycidyl ether fixation, or any combination thereof.

[0120] [Example 18] The method of example 14, further comprising optical tissue clearing.

[0121] [Example 19] The method of Example 14, wherein the combining step occurs after the incubating step.

[0122] [Example 20] The method of example 14, wherein the probe is provided with the chaotropic ion for co-diffusion into the tissue.

[0123] [Example 21] The method of example 14, wherein the probe is provided as a complex with the complexing agent to enhance mobility within the tissue.

[0124] References

[0125] Lai, HM, Tang, Y., Lau, ZYH, Campbell, RAA, Yau, JCN, Chan, DCW, Wong, HKT, Yan, LYC, Wu, WKK, Wong, SH, Kwok, KW, Wing, YK, Ng, HK, Mrsic-Flogel, TD, Mok, VCT, Chan, JYK, Ko, H. 2022. Antibody stabilization for thermally accelerated deep immunostaining. Nature Methods. 19: 1137.

[0126] Lee, K., Lai, H. M., Soerensen, M., Hui, E., Cho, W., Ma, V., Ho, J., Chang, R.C. 2020. Optimized Tissue Clearing Minimizes Distortion and Destruction During Tissue Delipidation. Neuropathol. Appl. Neurobiol. doi.org / 10.1111 / nan.12673.

[0127] Lai, H. M., Liu, A. K. L. L., Ng, H. H. M., Goldfinger, M. H., Chau, T. W., DeFelice, J., Tilley, B. S., Wong, W. M., Wu, W., Gentleman, S. M. 2018. Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues. Nat. Commun. 9: 1066.

[0128] Lai, H. M., Ng, W. L., Gentleman, S. M., Wu, W. 2017. Chemicals Probes for Visualizing Intact Animal and Human Brain Tissue. Cell Chem. Biol. 24: 659-672.

[0129] Liu, A. K. L., Lai, H. M., Chang, R. C., Gentleman, S. M. 2016. Free-of-acrylamide SDS-based Tissue Clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues. Neuropathol. Appl. Neurobiol. 43: 346-351.

[0130] Barba-Bon, A., Salluce, G., Lostale-Seijo, I., Assaf1, K. I., Hennig, A., Montenegro, J., Nau, W. M. 2022. Boron clusters as broadband membrane carriers. Nature 603: 637.

Claims

1. 1. A supramolecular histochemistry system for staining, comprising: at least one probe, at least one chaotropic ion, and optionally at least one complexing agent; A supramolecular histochemical system for staining, wherein said at least one probe achieves penetration throughout the tissue with uniform distribution within the tissue for realizing high specificity of tissue staining.

2. the at least one probe is a plurality of probes; the at least one chaotropic ion is one type of chaotropic ion; And / or optionally, the at least one complexing agent is a plurality of complexing agents, supramolecular histochemistry system for staining according to claim 1 .

3. 2. The supramolecular histochemical system for staining according to claim 1, wherein the chaotropic ion is a derivative of unsubstituted closo-borane with more than 9 atoms, an unsubstituted closo-carborane with more than 9 atoms, any mixture thereof, or any derivative thereof in which the boron or carbon atoms are independently substituted with hydrogen, fluorine, chlorine, bromine, iodine, a hydroxy group, an amino group, or an alkyl group.

4. 4. The supramolecular histochemical system for staining according to claim 3, wherein the chaotropic ion is closo-dodecaborate.

5. The supramolecular histochemistry system for staining according to claim 1, wherein the complexing agent comprises a cyclodextrin, a cucurbituril, a calixarene, a cyclophane, a cryptand, a cryptophane, a chemical derivative of any of these, an oligomer having an anion-associative repeating unit, or any mixture thereof.

6. 6. The supramolecular histochemistry system for staining according to claim 5, wherein the cyclodextrin is hexakis-β-glucopyranose (α-cyclodextrin), heptakis-β-glucopyranose (β-cyclodextrin), or octakis-β-glucopyranose (γ-cyclodextrin), wherein, optionally, one or more of the hydroxy groups are independently substituted with an amino group, a methoxy group, a (2-hydroxy)propoxy group, a sulfato group, a guanidino group, an acetato group, an acetamide group, an azido group, a bromo group, an iodo group, a chloro group, a toluenesulfonyl group, a thiol group, a succinyl group, a phosphato group, a 4-sulfatobutoxy group, a carboxymethoxy group, or a (2-aminoethyl)amino group.

7. 7. The supramolecular histochemical system for staining according to claim 6, wherein the cyclodextrin is (2-hydroxypropyl)-γ-cyclodextrin.

8. The supramolecular histochemistry system for staining according to claim 1 , wherein the plurality of probes comprises a primary antibody, an antigen, a lectin, a functionalized antibody, an oligopeptide, a small molecule dye, and / or a fluorescent DNA stain.

9. The supramolecular histochemical system for staining according to claim 1 , further comprising a chemical buffer system.

10. 10. The supramolecular histochemistry system for staining according to claim 9, wherein the chemical buffer system comprises Good's buffer, phosphate buffered saline (PBS), Tris, CAPS, HEPES, or any combination thereof.

11. 11. The supramolecular histochemical system for staining according to claim 10, wherein the chemical buffer system has a pH of 6 to 11 and a concentration of 10 to 1000 mM.

12. 10. The supramolecular histochemistry system for staining according to claim 9, wherein the chemical buffer system comprises 1x PBS at pH 7-8.

13. The supramolecular histochemical system for staining according to claim 1 , wherein the chaotropic ion and the complexing agent are provided in separate vehicles.

14. Providing a supramolecular histochemical system for staining according to claim 1; providing a specimen comprising tissue; incubating a specimen comprising the tissue with chaotropic ions of the supramolecular histochemical system to form an incubated specimen; combining the incubated sample with a system comprising a chemical buffer system and, optionally, a complexing agent of the supramolecular histochemical system to promote association of the probe with the target to generate a signal; imaging the signal; 1. A method for performing histology, comprising:

15. 15. The method of claim 14, wherein the combining step is dilution without a complexing agent.

16. 15. The method of claim 14, wherein the tissue-containing specimen is a tissue section having a thickness of about 1 to about 50,000 μm obtained by frozen sectioning, vibration sectioning, or paraffin sectioning, or is processed as an intact organ.

17. 15. The method of claim 14, wherein the tissue-containing specimen is fixed by formalin fixation, glutaraldehyde fixation, methanol fixation, ethanol fixation, glyoxal fixation, picric acid fixation, trichloroacetic acid fixation, polyacrylamide-formalin fixation, polyglycerol 3-polyglycidyl ether fixation, or any combination thereof.

18. 15. The method of claim 14, further comprising optical tissue clearing.

19. 15. The method of claim 14, wherein the combining step occurs after the incubation.

20. 15. The method of claim 14, wherein the probe is provided with the chaotropic ion for co-diffusion into the tissue.

21. The method of claim 14, wherein the probe is provided as a complex with the complexing agent to enhance mobility within the tissue.

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