Method for multiplex staining of sample

Abstract

The present disclosure provides a method for multiplex staining of a sample containing a target. In an embodiment, multiplex staining is performed by: using a plurality of target-binding means (e.g. antibodies) that bind to different epitopes of the same target; when the target is a sugar or sugar chain, using a plurality of sugar-chain-binding means (e.g. lectins) that have different sugar-chain-binding profiles, or, when the target is a nucleic acid, using a plurality of nucleic-acid-binding means (e.g. nucleic-acid-binding dyes) that have different nucleic-acid-binding profiles.

Description

Method for multiplex staining of samples

[0001] The present disclosure relates to a method for multiplex staining of samples. In one aspect, multiplex staining can be performed using a plurality of target-binding means (e.g., antibodies) that bind to different epitopes of the same target, a plurality of sugar chain-binding means (e.g., lectins) having different sugar chain-binding profiles when the target is a sugar or sugar chain, or a plurality of nucleic acid-binding means (e.g., nucleic acid-binding dyes) having different nucleic acid-binding profiles when the target is a nucleic acid.

[0002] Immunostaining of cells is commonly used in cell analysis and evaluation. Patent Document 1 discloses a method for co-staining actin using a plurality of different visualization means that bind to actin.

[0003] Japanese Patent Application Laid-Open No. 2023-25674

[0004] The present inventors have found that multiplex staining with a plurality of different antibodies that bind to different epitopes of the same target produces different staining images depending on the environment and state of existence of the target. This was surprising. The staining images are considered to reflect the environment and state of existence of the target, and presumably because the structure of the epitope changes in response to changes in the environment and state of existence of the target.

[0005] In the above Patent Document 1, paraffin-embedded samples could not be observed. However, the inventors have found that, for example, by using antibodies, it is possible to observe thin sections prepared from paraffin-embedded samples.

[0006] The inventors have further found that sugars and nucleic acids can be multiplex-stained using a plurality of detection means. In any of the multiplex stainings, cells and tissues did not stain uniformly and exhibited different colors for each cell or tissue.

[0007] The present disclosure provides the following inventions: (1) A method for staining a target, comprising: (i) multiple staining an isolated sample containing the target by applying a plurality of target-binding means (e.g., antibodies) that bind to different epitopes of the target; (ii) multiple staining the sample by applying a plurality of different target-binding means (e.g., lectins) that bind to the target, if the target is a sugar or sugar chain; or (iii) multiple staining the sample by applying a plurality of different target-binding means or nucleic acid-binding dyes that bind to the target, if the target is a nucleic acid. (2) The method according to (1), comprising: (i) multiple staining an isolated sample containing the target by applying a plurality of target-binding means that bind to different epitopes of the target. (3) The method according to (2), wherein the plurality of target-binding means comprises three or more types of target-binding means. (4) The method according to (3) or (4), wherein the plurality of target-binding means do not compete with any other target-binding means for binding to the same target. (5) The method according to (1), comprising applying (ii) a plurality of different target-binding means that bind to the target, wherein the target is a sugar or a sugar chain, to an isolated sample containing the target. (6) The method according to (1), comprising applying (iii) a plurality of different nucleic acid-binding dyes that bind to the target, wherein the target is a nucleic acid, to an isolated sample containing the target. (7) The method according to any one of (1) to (6), wherein the sample comprises a tissue section obtained by deparaffinizing a thin-layer section of a paraffin-embedded tissue block. (8) The method according to any one of (1) to (7), wherein the target is located in one or more regions selected from the group consisting of on the cell membrane, inside the cell, in the cytoplasm, inside the nucleus, and outside the cell. (9) A multiple staining kit for use in the method according to any one of (1) to (8), comprising a plurality of different target-binding means as defined in any one of (1) to (8). (10) A multiple stain image of the sample obtained by any of the methods described in (1) to (8) above. (11) A channel-resolved image, a single stain image, or a pseudo-single stain image obtained from the multiple stain image of (10) above.(12) A method for obtaining a combination of target-binding means including a plurality of target-binding means by staining a target, comprising: (a) providing a plurality of target-binding means that bind to different epitopes of the target; and (b) any of the following steps (i) to (iii): (i) multiple staining of the sample by contacting the plurality of target-binding means that bind to different epitopes of the target; (ii) multiple staining of the sample by contacting the sample with a plurality of different target-binding means that bind to the target, if the target is a sugar or sugar chain; or (iii) multiple staining of the sample by contacting the sample with a plurality of different target-binding means or nucleic acid-binding dyes that bind to the target, if the target is a nucleic acid, thereby obtaining a combination of target-binding means that visualize the target in different ways.

[0008] This demonstrates the significance of a technique for simultaneously staining the same target with multiple antibodies of different epitopes. Multiplex staining of the same target detects differences in the state of the target's existence, rather than the presence or quantity of the target. Depending on the state of the target, the antibody to which it binds most strongly varies. For example, each probe can detect the state of the target molecule (e.g., binding state with other molecules, physical deformation, physiological deformation, and differences in isoforms) with different sensitivities and specificities. This changes the image of the multiplex staining. The image shows the results of multiplex staining of cultured LLC cells and U2OS cells using three different anti-actin antibodies. It is shown that the overall color of the cells is completely different depending on the cell type. The image shows the results of multiplex staining of cultured LLC cells and U2OS cells using three different anti-actin antibodies. It is shown that the staining image differs depending on the antibody (more specifically, which parts of the cell's actin are stained differs). The image shows the results of multiplex staining of cultured U2OS cells using three different anti-tubulin antibodies. It has been shown that different antibodies produce different staining patterns (more specifically, different parts of the microtubules in the cell are stained). The results show that HeLa cells cultured under stressed and unstressed conditions were multiplexed using three different anti-TDP43 antibodies. It has been shown that different antibodies produce different staining patterns. Even under unstressed conditions (control), the three different anti-TDP43 antibodies produced different staining patterns, and under stressed conditions (stressed), the three different anti-TDP43 antibodies also produced different staining patterns, with each staining pattern change being unique. The results show that 10.5-day mouse embryo sections were multiplexed using three different anti-actin antibodies. It has been shown that different antibodies produce different staining patterns (more specifically, different parts of the actin in the embryo are stained). A method for detecting cells using multiple different glycan-binding methods is described. This method does not observe the environment or state of glycans, but rather observes differences in the types of glycans that differ from cell to cell. Because lectins have low specificity for glycans, one lectin can bind to multiple glycan structures.While a single lectin is not easily used for cell identification, using multiple lectins allows for more sensitive detection of cell differences by utilizing the differences in the binding spectrum of each lectin to sugar chains. The results show multiple staining of cultured HEK293 cells, RFL cells, and U2OS cells with different lectins. Each cell binds to a different lectin. The results of extracting defined color channels from multiple staining images obtained by multiple staining cultured HEK293 cells, RFL cells, and U2OS cells with different lectins are shown. The results show multiple staining of 10.5-day mouse embryo sections using three different lectins. It is clear that the staining image differs depending on the lectin. In the merged image, areas with strong red, strong purple, and strong green are displayed colorfully. The results of extracting defined color channels from multiple staining images obtained by multiple staining embryo sections with three different lectins are shown. The results show multiple staining of cultured U2OS cells and HEK293 cells using three different nucleic acid-binding dyes. The image shows how cells are stained in different colors. This is the result of multiple staining of a 10.5-day mouse embryo section using three different nucleic acid-binding dyes. It was clearly observed that some areas of the embryo were stained pink to reddish-purple, while others were stained green to blue.

[0009] <Definition of Terms> In this specification, “cell” means any cell, including prokaryotic cells and eukaryotic cells. Cells may be cells isolated from the environment (e.g., soil, water, air, biofilm, implant surface, and inside and on the surface of plants and animals). Cells may be isolated and cultured cells. Cells may be cloned cells. Cells may be contained in a sample isolated from the environment, and such a sample may contain multiple different cells. Cells may be present in isolated tissues of plants and animals. Cells may be contained in bodily fluids such as blood.

[0010] In this specification, “tissue” refers to a part of plants or animals, a collection of cells formed by the aggregation of multiple cells to perform a specific function. Tissue may be normal tissue or may include abnormal tissue (especially diseased tissue), such as cancer or inflammation. Examples of tissue, though not limited to, include epithelial tissue (e.g., epidermis and dermis of the skin, ciliated epithelium of the airways, absorptive epithelium of the intestines), connective tissue (e.g., adipose tissue, cartilage tissue, bone tissue), muscle tissue (e.g., skeletal muscle, cardiac muscle, smooth muscle), blood vessels, lymphatic vessels, and nervous tissue (e.g., brain, spinal cord, peripheral nerves). Tissue may be an organ or a part of an organ. Examples of organs or tissues include: digestive organs or tissues such as the mouth, salivary glands, pharynx, esophagus, stomach, small intestine, large intestine, rectum, liver, gallbladder, and pancreas; respiratory organs or tissues such as the nasal cavity, pharynx, larynx, trachea, bronchi, and lungs; circulatory organs or tissues such as the heart, blood vessels (especially arteries, veins, and capillaries); urinary organs or tissues such as the kidneys, ureters, bladder, and urethra; endocrine organs or tissues such as the pituitary gland, thyroid gland, parathyroid gland, adrenal gland, pancreas, and gonads (e.g., testes and ovaries); male reproductive organs or tissues such as the testes, epididymis, vas deferens, prostate gland, and penis; female reproductive organs or tissues such as the ovaries, fallopian tubes, uterus, and vagina; nervous organs or tissues such as the brain, spinal cord, and peripheral nerves; lymphatic organs or tissues such as lymph nodes, spleen, and thymus; and sensory organs such as the skin (scalp and other skin), eyes, ears, nose, and tongue.

[0011] In this specification, “Sample” refers to the subject of testing in this disclosure. A sample is, for example, a sample obtained from a living organism and includes, for example, cells or tissues. A sample may also be, for example, isolated from the environment. A sample may be a solid sample (e.g., solid tissue, cell aggregates, precipitates after centrifugation, etc.) or a liquid sample (e.g., bodily fluids such as blood, plasma, serum, saliva, urine, sweat, ascites, pleural fluid, cerebrospinal fluid, etc.). A sample may also be an animal embryo.

[0012] In this specification, “target” refers to the substance to be detected in this disclosure. A target may be, for example, a biomolecule, and may be, for example, a peptide (including proteins), nucleic acids, sugars, glycoproteins, extracellular matrix, and infectious organisms or pathogens such as viruses, bacteria, fungi, and parasites. A target may be located inside, on, or outside of a cell. A target may also be an exogenous substance. This is useful, for example, when evaluating how an exogenous target is distributed within a living organism.

[0013] In this specification, “isolate” means to separate from the environment (including living organisms). Isolation does not necessarily mean purification to a single substance. However, isolation does include purifying the target to a substantial single substance. Cell isolation means separating cells from other components and includes cell cloning. A subject that has been isolated and then mixed with other components is still an isolated subject, and the isolated subject is present in the mixture with other components.

[0014] In this specification, "multiple staining" refers to staining a sample containing a target, using multiple target-binding means having different binding affinities or binding profiles to the target, and distinguishing and identifying each target-binding means by distinctly different target detection means, while each target-binding means exhibits a distinctly different color; or the target-binding means themselves are distinctly different probes, and the target exhibits a distinctly different color for each target-binding means, thereby staining the sample. The target-binding means themselves may be affixed with distinctly different target detection means (e.g., probes), or the antibodies that distinguishly recognize the target-binding means may be affixed with distinctly different target detection means (e.g., probes). For example, in the case of multiple staining using antibodies, the antibodies themselves may be affixed with distinctly different probes, or the secondary antibodies that distinguishly recognize the antibody (primary antibody) may be affixed with distinctly different probes. Nucleic acid-binding means may themselves emit distinctly different signals (e.g., fluorescence). In this specification, "distinguishable" means that the target-binding means are observed as distinct from one another. Distinguishing properties are typically achieved by the target binding means being linked in a one-to-one correspondence with the target detection means (including cases where the target binding means itself also functions as the target detection means), and by the target detection means being observable separately as distinct entities. For example, if the target detection means is a fluorescent probe, a specific wavelength can be separated from other wavelengths using, for example, a dichroic mirror, a dichroic beam splitter, or a bandpass filter, thereby allowing fluorescence originating from a specific target binding means to be observed separately from fluorescence originating from other target binding means.

[0015] In this specification, “antibody” means immunoglobulin. Antibodies can be of various isotypes, for example, IgG. Antibodies may be polyclonal antibodies, but preferably monoclonal antibodies. Antibodies may, though not necessarily, be human chimeric antibodies, humanized antibodies, or human antibodies. Human chimeric antibodies can be produced by replacing the constant region of a non-human antibody with the constant region of a human antibody. Humanized antibodies can be produced by replacing the six CDRs of a human antibody with the six corresponding CDRs of a non-human antibody. Human antibodies can be produced using animals (e.g., mice) in which at least the heavy chain variable region of the immunoglobulin has been replaced with the corresponding region of a human gene locus. If the constant region is non-human, a human antibody can be obtained by replacing the constant region with the amino acid sequence of a human antibody. In this specification, antibodies are preferably humanized antibodies. In this specification, antibodies are preferably human antibodies. Antibodies have a signal peptide when produced intracellularly, but this signal peptide is excised when secreted extracellularly. Therefore, when administered as a medicine, the antibody does not require a signal peptide. The term antibody shall include antigen-binding fragments in addition to full-length antibodies (or non-antibody fragments).

[0016] In this specification, "CDR" refers to complementarity-determining regions located in the heavy chain variable region and the light chain variable region of an antibody. There are three CDRs in both the heavy chain and light chain variable regions, and they are referred to as CDR1, CDR2, and CDR3 from the N-terminus. CDRs can be determined, for example, based on the numbering by Kabat et al. (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.).

[0017] In this specification, "antigen-binding fragment of an antibody" means a fragment of an antibody that maintains its ability to bind to an antigen. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), diabody, sc(Fv)2 (single-chain (Fv)2), and links or complexes of two or more of these. For example, Fab can be obtained by digesting an antibody with papain. Alternatively, F(ab')2 can be obtained by digesting an antibody with pepsin, and Fab' can be obtained by further reduction of this. Other antigen-binding fragments of antibodies can also be prepared by methods well known to those skilled in the art. Such antigen-binding fragments of antibodies can be used in the present invention.

[0018] In this specification, “antigen-binding protein” means a protein that is a target-binding means and binds to a specific substance. Antigen-binding proteins may be modified proteins or non-natural proteins or their antigen-binding fragments. The antigen-binding protein is not particularly limited as long as it is a protein (e.g., a scaffold protein) whose binding affinity to the antigen can be conferred by modifying its amino acid sequence and which has a β-sheet structure or helix structure at the N-terminus or C-terminus. Examples include antibodies, antigen-binding fragments of antibodies (e.g., scFv), single-chain variable domains (VHH domains) of camel or llama heavy-chain antibodies (e.g., Nanobody®), scaffold proteins with bacterial albumin-binding domains as the basic skeleton (e.g., ABD®), scaffold proteins with the 10th domain of human fibronectin as the basic skeleton (e.g., Adnectin®, Fing®, or Monobody®), scaffold proteins with the Z domain of Staphylococcus aureus protein A as the basic skeleton (e.g., Affibody®), scaffold proteins with human γ-B-crystallin as the basic skeleton (e.g., Affibody®) Phosphorus (Affilin®), scaffold proteins with archaeal DNA-binding protein Sac7d as the basic framework (e.g., Affitin®), scaffold proteins with triple antiparallel helices as the basic framework (e.g., Alphabody®), scaffold proteins with human or insect lipocalin as the basic framework (e.g., Anticalin®), and armadillo protein as the basic framework Scaffold proteins such as armadillo repeat protein, scaffold proteins with human C-type lectin domain CTLD3 as the basic skeleton (e.g., Atrimer®), scaffold proteins with polymerized LDLR-A module as the basic skeleton (e.g., Avimer®), and scaffold proteins with human tenascin C Fn3 domain as the basic skeleton (e.g., Centyrin®).Scaffold proteins based on the SH3 domain of human Fyn tyrosine kinase (e.g., Fynomer®), and scaffold proteins based on the human BPTI / LACI-D1 / ITI-D2 / APPI domain (e.g., Knitz domain). Examples include scaffold proteins based on the OB shape of aspartyl-tRNA synthetase from bilobaculum elofilm (e.g., Obody®), scaffold proteins based on the 14th extracellular domain of human fibronectin III (e.g., Pronectin®), scaffold proteins based on the leucine-rich repeat module of the variable lymphocyte receptor of jawless organisms (e.g., Repebody®), scaffold proteins based on human ankyrin repeat protein (e.g., DARPin®), and scaffold proteins based on steroid A or cystatin (e.g., Affimer®) (Skrlec, K. et al., Trends in Biotechnology, Vol. 33). No. 7: 408-418, 2015). In the above-mentioned scaffold protein, the location of amino acids involved in antigen binding affinity is well known, and by selecting amino acids suitable for binding to the antigen using ribosome display or phage display, a scaffold protein with desired antigen-binding properties can be obtained. A scaffold protein is a protein that has a backbone similar in tertiary structure to the parent protein (the protein before modification) and has the function of maintaining a paratope (especially its three-dimensional shape). The basic backbone refers to the part of the scaffold protein other than the intended modification site (e.g., the paratope). A paratope refers to the part of an antigen-binding protein that binds to the antigen. Antigen-binding proteins can be appropriately obtained by those skilled in the art and used as a target binding means. Obtaining a target binding means suitable for staining is a well-known and conventional technique. In the present invention, it is not important to select a superior antibody,While the antibody itself can be a common one, it is important to use multiple antibodies with different binding properties.

[0019] In this specification, "fluorescent protein" refers to a protein that, when irradiated with short-wavelength electromagnetic waves, absorbs energy and excites electrons, and when these electrons return to their ground state, emits longer-wavelength electromagnetic waves. Examples of fluorescent proteins include fluorescent proteins that emit visible light and near-infrared fluorescent proteins that emit near-infrared light.

[0020] In this specification, "Affimer" is a protein having a biologically inactive and physically stable variant of stephin A or cystatin as its basic skeleton, and having antigen-binding sites in two loops presented on the same side from the four β-sheet structures of the basic skeleton. The amino acid sequence of the loop portion can be made diverse. By making the amino acid sequence of the loop portion diverse, it is possible to obtain affimers with binding affinity to various antigens by phage display. As the basic skeleton of an affimer, for example, a variant of stephin A having the amino acid sequence corresponding to SEQ ID NO: 1 disclosed in WO2009 / 136182 can be used, and a variant of stephin A in which the fourth glycine of stephin A may be replaced with arginine is a protein having heterologous amino acid sequences in two loops presented on the same side from the four β-sheet structures of the basic skeleton (for example, amino acid sites 46-54 and 67-84 of stephin A). The affimer has an α-helix structure at its N-terminus. Affimers may also be proteins that have a plant-derived cystatin as their basic backbone, and may have antigen-binding sites on two loops presented on the same side from the four β-sheet structures of the basic backbone. Examples of cystatin-based affimers include any one of the sequences SEQ ID NOs. 1 to 6 disclosed in WO2014 / 125290. The molecular weight of the affimer may be approximately 12 to 14 kDa.

[0021] In this invention, a "nanobody" is an antigen-binding protein based on the variable region domain of an antibody consisting solely of a heavy chain, as discovered in certain animals, in contrast to antibodies which consist of a heavy chain and a light chain. This antibody, consisting solely of a heavy chain, is commonly found in dromedary camels, Bactrian camels, llamas, and alpacas, and can bind to the antigen solely through the variable region domain of the heavy chain. In recent years, similar antibodies consisting solely of heavy chains have also been discovered in cartilaginous fish (such as sharks). The nanobody has three complementarity-determining regions (CDRs), and binds to the antigen through these three CDRs. The nanobody can be obtained by immunizing animals that produce the above-mentioned antibody consisting solely of a heavy chain with an antigen, isolating B cells from the immunized animals, obtaining a cDNA library containing the variable region, incorporating it into a phage display library using M13 phage, and screening it with the antigen.

[0022] In this specification, "scFv" is a single-chain antigen-binding protein in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody are linked by a flexible peptide linker. The heavy chain variable region and light chain variable region recognize the antigen, thereby maintaining the antigen specificity of the original antibody, while the two are connected by a flexible linker to promote the association between the heavy chain variable region and the light chain variable region. The flexible linker used can freely change its structure depending on the association state between scFv and the antigen, and for example, a glycine-rich sequence of about 15 amino acids (for example, serine may be inserted to ensure hydrophilicity) may be preferably used. As a flexible linker, for example, a GS linker having a repeating structure of a unit containing multiple Gs (for example, 3 or 4 Gs) and one S may be used, preferably a linker having the amino acid sequence -(GGGGGS)3- (Sequence ID 1). scFv can be obtained by selecting from a phage library in which the antigen-binding sites of scFv having a framework sequence (e.g., framework sequences of mammals such as humans) have been randomized, using the desired antigen-binding ability as an indicator.

[0023] In this specification, “DARPin” refers to an antigen-binding protein developed by Molecular Partners AG. DARPin is an artificial protein having ankyrin repeat units (typically 2 to 30 units), each repeat unit containing a skeletal residue and a target interaction residue (see, e.g., WO2002 / 020565). The ankyrin repeat unit has a common folding structure consisting of two antiparallel α-helices followed by a β-hairpin {where the β-pair pin has a loop that binds to the next repeat unit}. The stacking of ankyrin repeat units forms a curved structure in DARPin. Target interaction residues may be present in the β-hairpin and the exposed portion of the first α-helix of the ankyrin repeat unit. DARPin can be obtained by selection based on its binding affinity to antigens. DARPin can be obtained using methods such as phage display, ribosome display, and plasmid display.

[0024] In this specification, "monobody" is also called FingR or Adnectin, and is a scaffold protein whose basic skeleton is the 10th domain of human fibronectin (fibronectin type III domain). This domain has a structure similar to the variable domain of an antibody, namely, it has seven β-sheet structures that form a β-sandwich and three loops on both sides corresponding to three complementarity-determining regions. Antigen binding specificity can be modified by the amino acid sequences of loop BC between the second and third β-sheets, loop DE between the fourth and fifth β-sheets, and loop FG between the sixth and seventh β-sheets. Alternatively, it can be modified by the amino acid sequences of the third, fourth, sixth, and seventh β-sheets in addition to loop CD between the third and fourth β-sheets and loop FG between the sixth and seventh β-sheets.

[0025] <Method of the Disclosure> The Disclosure provides a method for staining a target. The Disclosure allows for the use of multiple different target-binding means (e.g., antibodies or antigen-binding proteins) for the same target. Alternatively, the Disclosure allows for the use of multiple different target-binding means for sugars or glycans, where the target is a sugar or glycan. Alternatively, the Disclosure allows for the use of multiple different target-binding means for nucleic acids.

[0026] The staining described herein can be performed in vitro.

[0027] In one embodiment, the present disclosure provides a method for staining a target, comprising: (i) multiple staining an isolated sample containing the target by contacting the sample with a plurality of target-binding means bound to different epitopes of the target; (ii) multiple staining the sample by contacting the sample with a plurality of different target-binding means bound to the target, provided the target is a sugar or sugar chain; or (iii) multiple staining the sample by contacting the sample with a plurality of different target-binding means (or nucleic acid staining means) bound to the target, provided the target is a nucleic acid. In (i), the plurality of target-binding means may each bind to a different epitope of the same target.

[0028] In (i), multiple target binding means may bind to the same target simultaneously. In (i), some of the multiple target binding means may bind to the same target simultaneously. If one target binding means and another target binding means compete for binding to the same target, which target binding means binds reflects the state of the target's existence. The method of this disclosure is effective in all cases.

[0029] Multiple target binding means include two or more different target binding means. In one embodiment, multiple target binding means include two to five different target binding means. Multiple target binding means include, for example, three different target binding means.

[0030] In a preferred embodiment, all of the target-binding means are non-natural proteins. In a preferred embodiment, the non-natural proteins may be monoclonal. Monoclonal means that the target-binding means are introduced into a protein-producing cell to produce multiple copies of the same target-binding means, thereby resulting in a composition containing a target-binding means consisting substantially of a single type of non-natural protein. One of the target-binding means may be polyclonal. Polyclonal means that the composition consists of a mixture of means that bind to various epitopes of the target. A polyclonal target-binding means typically consists of a mixture of means that bind to dozens or more, or even more than 100, different epitopes of the target. This polyclonal target-binding means can be considered as a single target-binding means and linked to a single target-detection means for observation. In a preferred embodiment, one of the target-binding means may be a polyclonal antibody and the others may be monoclonal antibodies. In a preferred embodiment, two of the target-binding means may be polyclonal antibodies and the others may be monoclonal antibodies.

[0031] Multiple (m) target-binding means preferably include multiple (n) target-binding means that bind to different epitopes of the same target {where m and n are natural numbers greater than or equal to 2, and m ≥ n}. The same target means that the targets are identical. If the targets cannot be distinguished at the substance level, then the targets of these target-binding means are not identical. Therefore, if the target includes modified and unmodified forms and attempts are made to distinguish and identify them using different target-binding means (especially two different target-binding means), then the targets of these target-binding means are not identical. Accordingly, the case in which the target includes modified and unmodified forms and attempts are made to distinguish and identify them using different target-binding means (especially two different target-binding means) is excluded from the act described in (i) above. Typically, the case in which attempts are made to distinguish and detect phosphorylated and unphosphorylated forms of a target using different target-binding means (especially two different target-binding means), or to distinguish and detect glycosylated and unmodified targets using different target-binding means (especially two different target-binding means), is excluded from the act described in (i) above. However, when n target binding means target the same substance, even if the remaining (m-n) or a portion (t) of the m target binding means stain the modified and unmodified forms of the target, or if the remaining (m-n) or a portion (t) of the m target binding means stain different targets, such actions may be included in the methods of the present disclosure {wherein t is a natural number greater than or equal to 2, and t < m-n}.

[0032] In one preferred embodiment, n is 2, 3, 4, or 5, preferably 3.

[0033] m can be set to the extent that distinguishable detection is possible, but a person skilled in the art can set it appropriately.

[0034] Multiple target binding means may each have different binding characteristics or binding profiles with respect to the target. Depending on the environment and state of the target, the multiple target binding means will exhibit a change in binding affinity that differs from the change in binding affinity of one or more other target binding means to the target (e.g., enhancement, attenuation, stronger or weaker enhancement, or stronger or weaker attenuation). This makes it possible to detect changes in the environment and state of the target using multiple target binding means.

[0035] Target detection means may be further applied to the target binding means. To clarify the presence or localization of a target, the target binding means itself may include a target detection means (e.g., a probe, preferably a fluorescent probe), or a target detection means that specifically binds to the target binding means (e.g., an antibody labeled with a probe, or, if the target binding means is an antibody, a secondary antibody labeled with a probe) may be used. In cases where the target binding means itself includes a target detection means, the target binding means itself may also function as the target detection means, or the target binding means and the target detection means may be bound together (e.g., covalently).

[0036] When antibodies are used as the target detection method, multiple antibodies bind to different epitopes on the same target. The species of the sample and the species of the antibodies may be the same, but preferably they are different. When antibodies used as the target detection method (i.e., primary antibodies) are detected by secondary antibodies, each primary antibody may be an antibody of a different species. This allows for the differentiation and staining of primary antibodies by using secondary antibodies that recognize antibodies in a species-specific manner.

[0037] Various well-known probes can be used as probes. While not particularly limited, examples of probes include fluorescent probes, enzyme probes, metal nanoparticle probes, biotin-streptavidin labeled probes (detected in combination with further labeling), tandem dyes, chemical tags and click chemistry labeling, fluorogen-activated probes, and halo-tag labeling. The label is linked to a target-binding means, thereby providing a means for linking a target detection means to it.

[0038] A probe as a target detection means may include, for example, a fluorescent probe. In some embodiments, a fluorescent probe is a fluorescent probe. Examples of fluorescent probes include those having fluorescence wavelengths of 400 nm to 760 nm, or those having fluorescence wavelengths in the near-infrared region of 760 nm or more, such as fluorescent probes that emit fluorescence in the near-ultraviolet, blue, green, red, and near-infrared regions. A fluorescent probe may be a low-molecular-weight compound.

[0039] In some embodiments, a plurality of different (e.g., two) target detection means may emit fluorescence in the following ways: near-ultraviolet and blue, near-ultraviolet and green, near-ultraviolet and red, near-ultraviolet and near-infrared, blue and green, blue and red, blue and near-infrared, green and red, green and near-infrared, and red and near-infrared.

[0040] In some embodiments, multiple (e.g., three) different target detection means may emit fluorescence in the following ways: near-ultraviolet, blue, and green; near-ultraviolet, blue, and red; near-ultraviolet, blue, green, and near-infrared; near-ultraviolet, green, and near-infrared; near-ultraviolet, red, and near-infrared; blue, green, and red; blue, green, and near-infrared; and green, red, and near-infrared. In some embodiments, three different target means may emit blue, green, and red fluorescence, respectively.

[0041] In one embodiment, a plurality of different (e.g., four) target detection means may emit fluorescence in the following ways: near-ultraviolet, blue, green, and red; near-ultraviolet, blue, green, and near-infrared; near-ultraviolet, blue, red, and near-infrared; and blue, green, red, and near-infrared.

[0042] Examples of fluorescent proteins include blue fluorescent proteins (e.g., BFP and its variants), green fluorescent proteins (e.g., GFP and its variants), and red fluorescent proteins (mCherry, mRFP, DsRed and their variants). Examples of near-ultraviolet fluorescent probe compounds include those with a maximum fluorescence wavelength of approximately 300 nm to less than 400 nm, specifically Alexa Fluor 350.

[0043] Examples of blue fluorescent probes include those with a maximum fluorescence wavelength of approximately 430 nm to less than 480 nm, specifically the Alexa Fluor 405, Cruz Fluor 405, and iFluor 405.

[0044] As green fluorescent probes, fluorescent probes with a maximum fluorescence wavelength of about 500 nm or more and less than 560 nm can be mentioned. Specifically, Alexa Fluor 488, Cruz Fluor 488, iFluor488, FITC, etc. can be mentioned.

[0045] As red fluorescent probes, fluorescent probes with a maximum fluorescence wavelength of about 610 nm or more and less than 750 nm can be mentioned. Specifically, Alexa Fluor 555, Alexa Fluor 594, Cruz Fluor 555, Cruz Fluor 594, iFluor 555, iFluor 594, Rhodamine, etc. can be mentioned.

[0046] As near-infrared fluorescent probes, fluorescent probes with a maximum fluorescence wavelength of about 760 nm or more and less than 800 nm can be mentioned. Specifically, Alexa Fluor 647, Alexa Fluor 700, Cy5, Cy5.5, etc. can be mentioned.

[0047] As fluorescent probes, also, for example, fluorescent probes of the Alexa Fluor series such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), PerCP (percyanine-chlorophyll tandem), Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 700; cyanine dyes such as Cy2, Cy3, Cy5, Cy7; rhodamine dyes such as rhodamine B, rhodamine 6G, Texas Red can be mentioned. As fluorescent probes, also, quantum dots, near-infrared fluorescent probes (Cy5.5 and Cy7) can be mentioned. Tandem dyes are probes using fluorescence resonance energy transfer (FRET). For example, PE-Cy5, or APC-Cy7 can be preferably used as tandem dyes.

[0048] Biotin-streptavidin labeling is used by linking additional labels to streptavidin. By utilizing the specific and high affinity between biotin and streptavidin, a target detection means is bound to a biotin-labeled target binding means to detect the presence or localization of the target.

[0049] Labeling by chemical tags and click chemistry is a labeling method in which a target binding means is linked to a chemical tag, and a target detection means is linked to the target binding means by click chemistry. Click chemistry occurs, for example, between an azide group and an alkyne group. Using this, the target binding means is linked to the target detection means to detect the presence or localization of the target.

[0050] Fluorogen-activating probes are techniques in which non-fluorescent molecules become fluorescent by binding to an antibody. Examples include FIAsH and ReAsH.

[0051] HaloTag is a protein labeling technology that can bind to various fluorescent ligands with cell membrane permeability. By linking HaloTag to a target binding means, the target binding means can be detected using a fluorescent ligand as a target detection means.

[0052] In order to detect the target binding means distinguishablely, it is preferable to use probes with maximum fluorescence wavelengths separated by 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, or 100 nm or more. Also, it is preferable to use a combination of probes that can distinguish and detect the target binding means by spectrally separating their maximum fluorescence wavelengths using a band-pass filter or dichroic mirror provided in a fluorescence detection means such as a fluorescence microscope.

[0053] In one aspect, the target binding means can be an antibody that binds to the target. In one aspect, the target is a sugar or sugar chain, and the target binding means is a means that binds to the sugar or sugar chain and can be, for example, a lectin. In these cases, a target detection means is used in combination for target detection.

[0054] In some embodiments, the target is a nucleic acid, and the target-binding means often also serves as a target-detection means. Examples of nucleic acid-binding dyes include 4',6-diamidino-2-phenylindole (DAPI), Heochst dyes (e.g., Heochst 33342, Heochst 33258), propidium element (PI), ethidium bromide (EtBr), acridine orange (AO), cyber green, gel green, and gel red.

[0055] In one embodiment, the present disclosure provides a method for staining a target, comprising: (i) multiple staining an isolated sample containing the target by applying a plurality of antibodies bound to different epitopes of the target; (ii) multiple staining the sample by applying a plurality of different lectins bound to the target, if the target is a sugar or sugar chain; or (iii) multiple staining the sample by applying a plurality of different nucleic acid-binding dyes bound to the target, if the target is a nucleic acid. In (i), the plurality of antibodies may bind to different epitopes of the same target.

[0056] In (i), multiple antibodies may bind to the same target simultaneously. In (i), some of the multiple antibodies may bind to the same target simultaneously. When one antibody and another antibody compete for binding to the same target, which antibody binds reflects the state of the target's presence. The methods of this disclosure are effective in both cases.

[0057] Antibodies bind to targets. Examples of targets include animal, plant, bacterial, fungal, and archaeal targets. Targets can be intracellular, extracellular, or on the cell membrane. Targets may be free or complexed with other components. Targets may be monomeric or polymeric. In this invention, cells may be fertilized eggs, pluripotent stem cells, stem cells, progenitor cells, somatic cells, and germ cells (e.g., oocytes). In this invention, targets may be non-cellular components in organisms, such as flagella (e.g., sperm or microbial flagella). According to this invention, targets may be intracellular scaffolds such as actin, microtubules, septins, and intermediate filaments. According to the present invention, the targets may be intracellular actin (e.g., α-actin, β-actin, and γ-actin), microtubule tubulin (e.g., α-tubulin, β-tubulin, and γ-tubulin), septin, and the intracellular scaffold such as intermediate filaments (e.g., keratin, GFAP (glial cell fibrous acidic protein), vimentin, neurofilament-M, neurofilament-L, neurofilament-H, α-internexin, lamin B1, lamin B2, lamin A). According to the present invention, the targets are not particularly limited, but include, for example, myosin (e.g., myosin I, myosin II, myosin III, myosin IV, myosin V, myosin VI, myosin VIII, and myosin IX, and their heavy or light chains), dynein (e.g., axonemal dynein and cytoplasmic dynein), troponin (cTnl, cTnT, etc.), kinesin (not particularly limited, but include, for example, KIF1A, KIF1Bα, KIF1Bβ, KIF1C, KIF2A, KIF3, KIF4, KIF5, KIF13B, KIF17, KIF26A, KIFC1, KIFC2, and KIFC3, and other kinesins belonging to the kinesin superfamily). According to the present invention, the target is not particularly limited, but examples include membrane proteins (e.g., receptor proteins, G proteins, ion transporters (ion channels, ion pumps), and transporter proteins).According to the present invention, the target is not particularly limited, but examples include BAR domain proteins (Amphiphysin, Endophyllin A1, FCHO2, Pacsin2, IRSp53) and ESCRT-III proteins (CHMP2A, CHMP2B, CHMP4A, CHMP4B, CHMP4C). In addition, the target is not limited to the above and can be various biomolecules (e.g., proteins, lipids, nucleic acids, hormones, and sugars). According to the present invention, the target is not particularly limited, but can be, for example, a lipid membrane or a lipid membrane domain {for example, a lipid-soluble fluorescent probe or a lipid-binding motif can be used}. According to the present invention, viscous sugars, amyloid, collagen fibers, etc. can also be targets.

[0058] According to the present invention, cellular components other than the intracytoskeleton may also be subjects of observation. Examples of targets include Aβ (e.g., Aβ1-42), α-synuclein, TPD43, tau, huntingtin, and their phosphorylated forms, as well as their abnormal accumulation and aggregates.

[0059] Furthermore, other targets include, for example, Wilmus Tumor 1 (WT-1), Human Carbonhydrate Antigen 125 (CA-125), Carcinoembryonic Antigen (CEA), Human Telomerase Reverse Transcriptase (hTERT), Mucin-1 (Muc-1), Mucin-2 (Muc-2), Cancer / Testis Antigen 1B (CTAG1B / NY-ESO-1), Prostatic Acid Phosphatase (PAP), Prostate Specific Antigen (PSA), and Prostate Examples include Specific Membrane Antigen (PSMA), Survivin b, mutant ras, mutant p53, CD20, vascular epithelial growth factor (VEGF), E-cadherin, and anaplastic lymphoma kinase (ALK).

[0060] In some embodiments, the target-binding means comprises an antigen-binding protein, which does not include one, two, or all of the following selected from the group consisting of phalloidin, jasplaquinolide, and 9-[2-(hydroxymethyl)phenyl]-6-[(2,2,2-trifluoroethyl)iminio]xanthene-3-olate (HMRef) (see Nat Commun. 2015, 6: 6463). In some embodiments, the target-binding means comprises an antigen-binding protein, which does not include any actin-binding protein other than an antibody. In some embodiments, the multiple target-binding means comprises multiple (two, three, or more) different anti-actin antibodies, for example, all of which are antibodies.

[0061] Lectins are not particularly limited, but examples include C-type lectins (e.g., mannose-binding lectin (MBL), dexins (e.g., dexin-1, dexin-2), DC-SIGN), galectins (e.g., galectin-1, galectin-3, galectin-7), P-type lectins (e.g., mannose-6-phosphate receptor (e.g., M6PR), I-type lectins (e.g., Siglec, NCAM), L-type lectins (e.g., lysine, concanavalin A), and chitin-binding lectins (e.g., wheat germ agglutinin (WGA), hebecactus lectin (HCC)).

[0062] Examples of lectins include Aleulia aurantialectin (AAL), Bauhinia purpurealectin (BPL), concanavalin A (Con A), Datura stramonium lectin (DSL), Dolicos biflorus aglutinin (DBA), Erythrina cristagalilectin (ECL), Galanthus nivalis lectin (GNL), Griffonia simplicifolialectin I (GSL I), Jacarina lenticulae aglutinin (LCA), Lotus tetragonolobus lectin (LTL), Lycopersicon esculentum (tomato) lectin (LEL), Maacchia amurensis lectin I (MAL I), Maacchia amurensis lectin II (MAL II), Macula pomifera lectin (MPL), Narcissus pseudonarcissus lectin (NPL, NPA), Examples include DL), peanut aglutinin (PNA), Pheceolus vulgaris erythroaglutinin (PHA-E), Pheceolus vulgaris leucoaglutinin (PHA-L), Pisum sativum aglutinin (PSA), Lycinus communis aglutinin I (RCA I, RCA120), Sambucus nigra lectin (SNA, EBL), Solanum tuberosum (potato) lectin (STL, PL), soybean aglutinin (SBA), wheat germ aglutinin (WGA), succinylated wheat germ aglutinin (sWGA), Wisteria floribunda aglutinin (WFA, WFL), Ulex europeus aglutinin I (UEA I), and Bichia bilosalectin (VVL / VVA).

[0063] These methods may further include obtaining a stained image of a sample from a multiple-stained sample, and obtaining a multiple-stained image of a sample.

[0064] The target detection means used in the methods of this disclosure may preferably all be fluorescent dyes (which may also be fluorescent proteins).

[0065] Stained images can be obtained by observing a multi-stained sample using an image acquisition device, transferred to a computer, and stored in the computer's memory. The image acquisition device can be a conventionally known fluorescence detection method, such as observation using a fluorescence microscope, a fluorescence stereomicroscope, a confocal microscope, or a multiphoton microscope. The images stored in the memory can be processed on the computer.

[0066] For example, acquired stained images can be imported into a computer as different channels for each different probe, and then superimposed on the computer to obtain a superimposed image (merged image) from the stained images. The generation of the superimposed image (merged image) can be performed by appropriately selecting image analysis software such as ImageJ or Photoshop. The superimposed image (merged image) is a multi-stained image. Images of one or more or all of the channels selected from the group consisting of red (R), blue (B), green (G), cyan (C), magenta (M), and yellow (Y) may be extracted from the superimposed image (merged image). The image obtained in this way is called a single-stained image or a channel-resolved image. An image obtained by emphasizing only the signal of a specific stain, even though it is actually multi-stained, is called a pseudo-single-stained image. Methods for generating these images are also provided in this disclosure.

[0067] The methods of this disclosure may further include detecting differences in staining patterns due to differences in target binding means from multiple staining images, channel-resolved images, single staining images, and pseudo-single staining images. Differences in staining patterns suggest differences in the state of the target. Therefore, differences in staining patterns suggest differences in the state of the target, the environment in which the target exists, the cells or tissues containing the target, or the state of the cells or tissues containing the target.

[0068] The methods of the present disclosure may further include characterizing the state of a target, cell or tissue, or the state of a cell or tissue based on differences in staining patterns. For example, one cell may be characterized by staining around the cell membrane, while another cell may be characterized by staining throughout the cytoplasm. Alternatively, a cell may be characterized such that one part of the cell is primarily bound by target binding means A, while another part is primarily bound by target binding means B. The methods of the present disclosure may further include examining the state of a target, cell or tissue, or the state of a cell or tissue based on differences in staining patterns between target binding means. The methods of the present disclosure may also further include examining the state of a target, cell or tissue, or the state of a cell or tissue based on differences in staining patterns by the same target binding means.

[0069] By training machine learning or deep learning (AI) with training data that includes the relationship between staining patterns and cell types, and the relationship between staining patterns and cell states, and obtaining a trained model, it becomes possible to predict the type and state of cells and tissues from staining patterns. Furthermore, using the trained model, it becomes possible to predict the distribution of cells in tissues and the state of each cell.

[0070] Accordingly, the present disclosure provides a method for generating training data images for learning a discrimination mechanism that discriminates for determining the type or state of cells or tissues in a target multiple stained image, wherein, when a target multiple stained image is input, the method includes: (i) multiple staining the sample by contacting it with a plurality of target-binding means that bind to different epitopes of the target; (ii) multiple staining the sample by contacting it with a plurality of different target-binding means that bind to the target, if the target is a sugar or sugar chain; or (iii) multiple staining the sample by contacting it with a plurality of different target-binding means (or nucleic acid staining means) that bind to the target, if the target is a nucleic acid.

[0071] Furthermore, the Disclosure provides a method for examining cells or tissues, further comprising inputting multiple staining images of the cells or tissues into the trained model to predict the type and / or state of the isolated cells or tissues. The Disclosure also provides a trained model used in this method.

[0072] This disclosure provides servers, clients, computers, etc., that are equipped with the above-mentioned trained model and perform the above-mentioned testing method.

[0073] <Staining Kit of the Disclosure> The Disclosure provides a staining kit for use in carrying out the method of the Disclosure. The Disclosure provides the use of a target binding means (or target detection means) for manufacturing a staining kit for use in carrying out the method of the Disclosure.

[0074] The staining kit of this disclosure includes a target binding means. The staining kit of this disclosure further includes a target detection means. The target binding means and the target detection means are as described above, so please refer to the above and do not repeat them here.

[0075] <Multi-stained and channel-resolved images of the Disclosure> The method of the Disclosure provides a multiple-stained image of a sample. Channel-resolved images, single-stained images, and pseudo-single-stained images can also be obtained from the multiple-stained image. Therefore, the Disclosure provides a multiple-stained image of a sample, as well as channel-resolved images, single-stained images, and pseudo-single-stained images. These images can be communicated (sent and received) between computers over a network (e.g., the Internet). These images can be recorded on storage devices with non-volatile memory (e.g., computers, USB memory, magnetic tape, SSD, HDD, CD-R, and DVD-R).

[0076] Example 1: Staining of the same target with multiple antibodies with different epitopes

[0077] (1) Multiple antibodies targeting different protein paint epitopes were prepared, and different probes were attached to each, followed by cell staining (see Figure 1). Figure 1 shows that when there are differences in the amount of each antibody bound to each epitope, the staining of the target (specifically, the color) changes.

[0078] Sample fixation and staining were performed as follows: 1. Samples were treated with a fixative (1x D-PBS(-) Fujifilm 048-29805, 4% formaldehyde Fujifilm 064-0041) for 20 minutes. This yielded the following: Formalin-fixed organoids, frozen sections, tissue sections, slides, deparaffinized tissue sections, slides (0.1% Triton X-100, untreated). 2. Samples were washed three times with 1x D-PBS(-). *1 mM MgCl2 (actin) or 1 mM EGTA (microtubules) were added depending on the target. 3. Samples were allowed to stand in a permeabilization solution (Triton X-100 Fujifilm A16046, 1x D-PBS(-) PBS) at room temperature for 5–10 minutes to remove the cell membrane. *1 mM MgCl2 (actin) or 1 mM EGTA (microtubules) were added depending on the target. 4. The sample was treated with a blocking solution (0.2% fish collagen (Nippi), 1x D-PBS(-)). 5. The sample was reacted with the primary antibody. Can Get Signal(trademark) Solution 1 TOYOBO NKB-201 was used as the antibody diluent. The antibody reaction time was 1 hour to overnight. 6. The sample was washed three times with 1x D-PBS(-). 7. The sample was reacted with the secondary antibody. Can Get Signal(trademark) Solution 1 TOYOBO NKB-201 was used as the antibody diluent. The antibody reaction time was 1 hour to overnight. 8. The sample was washed three times with 1x D-PBS(-). 9. The sample was treated with 4% formaldehyde for 20 minutes and re-fixed. 10. The sample was washed three times with PBS. 11. Subsequently, the sample was subjected to microscopic observation, and the obtained images were imported into a computer to create a merged image.

[0079] Example 1-1: Staining with anti-actin antibodies LLC cells and U2OS cells were cultured on different plates, and merged images of cell staining with three fluorescently labeled anti-actin antibodies that bind to different epitopes were obtained. The actin antibodies used were Beta Actin Polyoclonal antibody (proteintech 20536-1-AP Rabbit IgG), Anti Actin antibody (millipore MAB1501 mouse IgG1), and Anti-β-Actin antibody (Sigma A2228 mouse IgG2a). Thus, one polyclonal antibody and two monoclonal antibodies were used.

[0080] The results are shown in Figure 2. Figure 2 reveals that LLC cells and U2OS cells exhibit completely different staining patterns. In LLC cells, the cell edges tended to stain orange, and the cytoplasm tended to stain green. In contrast, in U2OS cells, the entire cell was stained pink or purple, and some actin fibers were stained green. Thus, it became clear that even when staining the same target, different parts of the same cell were stained with different colors.

[0081] Figure 3 shows the staining images of each channel when LLC cells and U2OS cells are stained. It demonstrates that different antibodies stain different regions of actin filaments within the cells.

[0082] The results suggest that the difference in surface state of actin structures is due to varying locations, and that the affinity of each antibody, which recognizes different epitopes, to these actin structures also varies. As mentioned above, the staining patterns differed significantly depending on the cell type. Therefore, this method could potentially be used for selective staining of cells.

[0083] Examples 1-2: Staining with anti-tubulin antibodies U2OS cells were cultured on a plate, and merged images of cell staining with three fluorescently labeled anti-tubulin antibodies that bind to different epitopes were obtained by the above staining method. Alpha Tubulin Polyclonal antibody (proteintech1224-1-AP) was used as the first primary antibody, and Goat Anti Rabbit IgG HL Alexa FluorR 405 was used as the secondary antibody to detect it. Monoclonal Anti-α-Tubulin antibody produced in mouse (Sigma T-9026) was used as the second primary antibody, and Goat Anti Mouse IgG1 (g1) Alexa FluorR 488 was used as the secondary antibody to detect it. Alpha Tubulin Monoclonal antibody (Proteintech 66031) was used as the third primary antibody, and Goat Anti Mouse IgG2b (g2b) Alexa FluorR 594 was used as the secondary antibody to detect it.

[0084] The results are shown in Figure 4. Figure 4 reveals that the periphery of the cell was stained green, while the inside of the cell was stained yellow or orange. The antibodies are thought to be staining the microtubules inside the cell. It can be understood that the differences in the state of microtubules within the cell are differentiated by the staining of these three antibodies, each possessing a different epitope.

[0085] Examples 1-3: Staining with anti-TDP43 antibodies HeLa cells were cultured on plates, and merged images of cell staining with three fluorescently labeled anti-TDP43 antibodies that bind to different epitopes were obtained by the above staining method. The cells were cultured under conditions with and without stress. Stress was applied by treatment with 0.5 mM sorbitol.

[0086] The results are shown in Figure 5. Figure 5 shows that each anti-TDP43 antibody showed different staining patterns on cells cultured in the absence of stress. The staining patterns of each antibody were different on cells cultured in the presence of stress, but the staining patterns changed significantly from those in the absence of stress.

[0087] In Example 1-1, an anti-actin antibody was used, and in Example 1-2, an anti-tubulin antibody was used. This suggested that intracellular targets can be selectively stained according to their state using antibodies that bind to different epitopes of the same target, and that this can be used to selectively stain different cells. In Example 1-3, the staining method of the present invention was applied to proteins other than the cytoskeleton. These experiments suggested that a scheme of staining the same target using antibodies that bind to different epitopes is useful as a means of detecting the type and state of cells.

[0088] Furthermore, none of the antibodies produced matching staining patterns, clearly indicating that antibodies fundamentally produce different staining patterns depending on the epitope. Therefore, staining the same sample results in differences in staining patterns due to the antibodies, leading to differences in stained and unstained areas in the merged image. In this example, three types of antibodies were used, but any two of them could be used to selectively stain according to the presence or absence of the target.

[0089] Examples 1-4: Embryo staining with different actin antibodies. In the previous examples, cultured cells were the target of staining. In this example, sections of 10.5-day-old mouse embryos were subjected to the staining of the present invention. The same antibody set described in Example 1-1 was used as the antibody.

[0090] The results are shown in Figure 6. Figure 6 suggests that different antibodies stain different parts of the embryo. As a result, the embryo was stained in a variety of colors in the merged image. This method is suggested to be useful in detecting abnormal parts of the embryo.

[0091] Japanese Patent Publication No. 2023-25674 disclosed a method for multiple staining actin using different binding means. However, with this method, paraffin-embedded samples were difficult to stain after deparaffinization due to the influence of the organic solvent used during the treatment. According to this embodiment, good multiple staining was possible even for deparaffinized samples when an antibody was used.

[0092] Example 2: Staining of the same cell or tissue with multiple different glycan probes (2) Glycan paint Since the types and combinations of glycans present on the cell surface differ from cell to cell, cells can be stained with different colors depending on the type by staining with glycan probes that have different fluorescent labels (see Figure 7). In glycan paint, lectins show different binding profiles to glycans, so it is not the same glycan being stained with different lectins, but rather the differences in the glycan expression profiles of cells are stained, which is different from Example 1.

[0093] The samples were fixed and stained as follows: 1. The samples were fixed by treating them in fixative (1x D-PBS(-), 4% formaldehyde) for 20 minutes. This yielded the following: Deparaffinized tissue section slides, frozen sections, tissue section slides, formalin-fixed organoids. 2. The samples were washed three times with washing solution (1x D-PBS(-), 1 mM MgCl2, 0.1 mM MnCl2, 1 mM CaCl2). 3. Glycan staining of the samples was performed by reacting the samples with 0.7 μL each of ConA 405* / wheat germ agglutinin 488 / tomato Lectin 594 in PBS. The reaction was carried out for 1 hour to overnight. 4. The samples were washed three times with washing solution (1x D-PBS(-), 1 mM MgCl2, 0.1 mM MnCl2, 1 mM CaCl2). 5. The sample was treated with 4% formaldehyde for 20 minutes and re-fixed. 6. The sample was washed three times with a washing solution (1x D-PBS(-), 1 mM MgCl2, 0.1 mM MnCl2, 1 mM CaCl2). 7. The sample was then subjected to microscopic observation, and the obtained images were imported into a computer to create a merged image.

[0094] HEK293 cells, RFL cells, and U2OS cells were cultured and subjected to the lectin staining method described above. The staining results are shown in Figures 8 and 9. As shown in Figure 8, the reactivity of each lectin to each cell type differed, and this difference resulted in significant differences in the staining results in the merged images. HEK293 cells reacted strongly to wheat germ agglutinin and emitted green fluorescence overall, while U2OS cells reacted strongly to lectin 59A and emitted red fluorescence. Figure 9 shows the results of applying filters to the images obtained above to extract each color. Here again, it is clear that the color staining differs depending on the cell type.

[0095] Next, 10.5-day mouse embryos were stained using the same three lectins. The results are shown in Figures 10 and 11. As shown in Figures 10 and 11, the areas stained by each lectin differed, resulting in an uneven appearance with red, blue, and green areas. Figure 11 shows the results of applying filters to the images obtained above to extract each color. Here again, it is clear that different parts of the embryo were stained in different colors.

[0096] Unlike antibodies, lectins do not stain the same glycan chain, but multiple staining was possible even when using multiple lectins.

[0097] Example 3: Nucleic acid paint staining of the same cells or tissue with multiple different nucleic acid probes 1. The sample was fixed by treatment in fixative (1x D-PBS(-), 4% formaldehyde) for 20 minutes. This yielded the following: Deparaffinized tissue section slide Frozen section Tissue section slide Formalin-fixed organoid 2. The sample was washed three times with 1x D-PBS(-). 3. The sample was subjected to nucleic acid staining. Cellstain™-PI solution, Cellstain™-Hoechst 33342 solution, Cellstain™-AO solution (Dojin Chemical Co., Ltd.) were used as staining agents. Staining was performed according to the manufacturer's manual, with treatment times ranging from 1 hour to overnight. 4. The sample was washed three times with 1x D-PBS(-). 5. The sample was treated with 4% formaldehyde for 20 minutes and re-fixed. 6. The sample was washed three times with 1x D-PBS(-). 7. The sample was then subjected to microscopic observation, and the obtained images were imported into a computer to create a merged image.

[0098] When U2OS cells and HEK293 cells were cultured and subjected to the above staining, differences in staining intensity were observed depending on the cell type, as shown in the left and right panels of Figure 12. HEK293 cells exhibited fluorescence between white and purple, while U2OS cells and RFL cells exhibited light red and dark red fluorescence, respectively. When the above staining was applied to embryos, the results are shown in Figure 13. As shown in Figure 13, the left center of the embryo exhibited reddish-purple fluorescence, while the right center to the upper right of the embryo exhibited blue-green fluorescence. In this way, multicolor imaging was possible by subjecting embryos to multiple nucleic acid staining.

Claims

1. A method for staining a target, comprising: (i) multiple staining an isolated sample containing the target by contacting the sample with a plurality of target-binding means bound to different epitopes of the target; (ii) multiple staining the sample by contacting the sample with a plurality of different target-binding means bound to the target, provided the target is a sugar or sugar chain; or (iii) multiple staining the sample by contacting the sample with a plurality of different target-binding means or nucleic acid-binding dyes bound to the target, provided the target is a nucleic acid.

2. The method according to claim 1, comprising (i) contacting an isolated sample containing the target with a plurality of target-binding means that bind to different epitopes of the target to perform multiple staining of the sample.

3. The method according to claim 2, wherein the plurality of target binding means include three or more types of target binding means.

4. The method according to claim 2 or 3, wherein the multiple target binding means do not compete with any other target binding means for binding to the same target.

5. The method according to claim 1, comprising (ii) multiple staining of an isolated sample containing the target by contacting the sample with a plurality of different target-binding means, wherein the target is a sugar or a sugar chain and is bound to the target.

6. The method according to claim 1, comprising (iii) multiple staining of an isolated sample containing the target by contacting the isolated sample with a plurality of different nucleic acid-binding dyes, wherein the target is a nucleic acid and binds to the target.

7. The method according to any one of claims 1 to 6, wherein the sample includes tissue sections obtained by deparaffinizing thin-layer sections of paraffin-embedded tissue blocks.

8. The method according to any one of claims 1 to 7, wherein the target is located in one or more regions selected from the group consisting of the cell membrane, inside the cell, inside the cytoplasm, inside the nucleus, and outside the cell.

9. A multiplex staining kit for use in the method according to any one of claims 1 to 8, comprising a plurality of different target binding means as defined in any one of claims 1 to 8.

10. A multiple stain image of a sample obtained by the method described in any one of claims 1 to 8.

11. A channel-resolved image, a single-stained image, or a pseudo-single-stained image obtained from a multiple-stained image according to claim 10.

12. A method for obtaining a combination of target-binding means including a plurality of target-binding means by staining a target, comprising: (a) providing a plurality of target-binding means that bind to different epitopes of the target; and (b) any of the following steps (i) to (iii): (i) multiple staining the sample by contacting the plurality of target-binding means that bind to different epitopes of the target; (ii) multiple staining the sample by contacting the sample with a plurality of different target-binding means that bind to the target, provided that the target is a sugar or sugar chain; or (iii) multiple staining the sample by contacting the sample with a plurality of different target-binding means or nucleic acid-binding dyes that bind to the target, provided that the target is a nucleic acid, thereby obtaining a combination of multiple target-binding means that visualize the target in different ways.

Images