JP2018162986A - Coloring method, coloring material and coloring kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a coloring method which colors a substance which includes an aldehyde group contained in a cell or a tissue by employing a low molecular weight compound capable of penetrating in the cell easier than an antibody and simultaneously is capable of performing well-known coloring such as HE coloring arbitrarily about an identical cell or tissue.SOLUTION: The coloring method includes [a] a coloring process X which colors the cell or tissue by stain solution containing one or more compounds selected from a group of the compound represented by a specified chemical formula and colors the substance including the aldehyde group contained in the cell or tissue. The coloring method includes [b] an oxidation treatment process which alters at least one of the hydroxy group of polysaccharide contained in the cell or tissue to the aldehyde group by performing the oxidation treatment of the cell or tissue in advance. The coloring method includes [c] a coloring process H which colors the basic substance contained in the cell or tissue. The coloring method further includes [d] a coloring process E which colors an acid substance contained in the cell or tissue.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a staining method, a staining agent, and a staining kit.

  Conventionally, hematoxylin-eosin staining (HE staining) has been performed when observing a tissue section with an optical microscope in a medical field such as basic research such as histology and pathology and pathological diagnosis. Hematoxylin has the property of staining basic cell nuclei blue-purple, and eosin has the property of staining acidic cytoplasmic components, endocrine granules, etc. in red pink. Utilizing these properties, information on cell morphology and lesions can be obtained from tissue samples stained with HE.

In addition, tissue sections may be immunostained by immunohistochemistry (IHC) using an antibody that can bind to a specific cell component (antigen).
Patent Document 1 proposes a tissue staining method in which HE staining and immunostaining are performed on the same tissue section to obtain information on the morphology of the tissue and information on specific cell components.

International Publication No. 2013/035688

  An antibody that binds to an antigen by immunostaining is a protein molecule that is larger than the size of the antigen, and has a problem that it is difficult to reach the antigen inside the cell through the cell membrane. For this reason, the cell membrane is destroyed by treating the tissue section with alcohol, and a treatment for allowing the antibody to enter the cell is performed. However, there is a concern about the morphological change of the tissue accompanying the destruction of the cell membrane, and it is difficult to achieve both HE staining and immunostaining.

  The present invention stains a substance having an aldehyde group contained in a cell or tissue using a low molecular weight compound that can penetrate into cells more easily than an antibody, and optionally stains the same cell or tissue with HE. A staining method capable of performing known staining such as the above is provided. In addition, a staining agent and a staining kit used in the staining method are provided.

[1] An aldehyde group contained in the cell or tissue by staining the cell or tissue with a staining solution containing one or more compounds selected from the group of compounds represented by the following formulas (1) to (5) The dyeing method which has the dyeing | staining process X which dye | stains the substance which has this.

[In formula (1), at least one of six hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 6. , A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (2), at least one of the 10 hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 10, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (3), at least one of the eight hydrogen atoms is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 8, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (4), the four hydrogen atoms at the 3, 4, 5, 6 positions of the diaminobenzene structure may be substituted with an arbitrary substituent, and the hydrogen atoms at the 3, 4 positions are removed to form carbon. The cycloalkane Q of formula 4-7 may be condensed, and one or more methylene groups constituting the cycloalkane Q are a (—O—) group or (— Optionally substituted by an NH-) group;
In formula (5), at least one of the three hydrogen atoms of benzofurazan is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 3 And a hydrogen atom not substituted with R may be substituted with any substituent. ]
[2] The staining according to [1], including an oxidation treatment step in which at least a part of hydroxyl groups of the polysaccharide contained in the cells or tissues is changed to aldehyde groups by oxidizing the cells or tissues in advance. Method.
[3] The staining method according to [1] or [2], which includes a staining step H for staining a basic substance contained in the cell or tissue before or after the staining step X.
[4] The staining method according to [3], wherein in the staining step H, the basic substance is stained with a staining solution containing hematoxylin.
[5] The staining according to [1] or [2], wherein the staining step X and the staining step H for staining a basic substance contained in the cell or tissue are simultaneously performed on the cell or tissue. Method.
[6] The staining method according to [5], wherein a staining solution containing at least one compound selected from the group of compounds represented by the formulas (1) to (5) and hematoxylin is used.
[7] Any of [3] to [6], further comprising a staining step E for staining the acidic substance contained in the cells or tissues with respect to the cells or tissues that have passed through the staining step X and the staining step H The staining method according to claim 1.
[8] The staining method according to [7], wherein, in the staining step E, the acidic substance is stained with a staining solution containing eosin.
[9] The staining method according to [1], including the staining step X and a step of performing HE staining on the same tissue section.
[10] The staining method according to [1], comprising the staining step X and a step of performing Papanicolaou staining on the same cell specimen.
[11] The staining method according to [1], including the staining step X and a step of performing Giemsa staining on the same blood specimen.
[12] A staining agent comprising one or more compounds selected from the group of compounds represented by the following formulas (1) to (5).

[In formula (1), at least one of six hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 6. , A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (2), at least one of the 10 hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 10, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (3), at least one of the eight hydrogen atoms is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 8, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (4), the four hydrogen atoms at the 3, 4, 5, 6 positions of the diaminobenzene structure may be substituted with an arbitrary substituent, and the hydrogen atoms at the 3, 4 positions are removed to form carbon. The cycloalkane Q of formula 4-7 may be condensed, and one or more methylene groups constituting the cycloalkane Q are a (—O—) group or (— Optionally substituted by an NH-) group;
In formula (5), at least one of the three hydrogen atoms of benzofurazan is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 3 And a hydrogen atom not substituted with R may be substituted with any substituent. ]
[13] The staining agent according to [12], further comprising hematoxylin.
[14] A staining kit comprising the staining agent according to [12] or [13].
[15] The staining kit according to [14], comprising the staining agent and a staining agent containing hematoxylin.
[16] The staining kit according to [14] or [15], further including a staining agent containing eosin.

  According to the staining method of the present invention, the compound represented by the formulas (1) to (5) is bound to a substance having an aldehyde group contained in a cell or tissue to be stained, and is derived from the compound. By observing blue fluorescence, information on the substance in the cell or tissue can be obtained. In addition, both the staining method of the present invention and conventional staining methods such as HE staining, Papanicolaou staining, Giemsa staining, and the like can be performed on the same cells or the same tissues. Thereby, for example, information on the tissue morphology by HE staining and information on the substance by the blue fluorescence observation can be obtained from the same tissue section. In other words, since two types of staining that were conventionally performed individually for two different tissue sections can be performed simultaneously for two types of staining for the same tissue section, pathological diagnosis can be performed more quickly and more easily. Can be implemented accurately.

It is the flowchart which showed an example of the order of the process and process in the dyeing | staining method of this invention. It is the fluorescence image which observed the tissue section dye | stained in Example 1 with the fluorescence microscope. 4 is a fluorescence image obtained by observing the tissue section stained in Example 2 with a fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 3 with the fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 4 with the optical microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 4 with the fluorescence microscope. 4 is a fluorescence image of a tissue section stained in Example 4 observed with a fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 5 with the optical microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 5 with the fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 6 with the optical microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 6 with the fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 7 with the optical microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 7 with the fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 8 with the optical microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 8 with the fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 9 with the optical microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 9 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 10 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 10 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 11 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 11 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 12 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 12 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 13 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 13 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 14 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 14 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 15 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 15 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 16 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 16 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 17 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 17 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 18 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 19 with the fluorescence microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 19 with the fluorescence microscope. It is the transmission image which observed the tissue section dye | stained in Example 20 with the microscope. It is the fluorescence image which observed the tissue section dye | stained in Example 20 with the fluorescence microscope. In a comparative experiment, it is the transmission image which observed the tissue section which carried out PAS dyeing | staining with the optical microscope. In a comparative experiment, it is the fluorescence image which observed the tissue section dye | stained by CHO with the fluorescence microscope.

《Dyeing target》
The cell or tissue to be stained by the staining method of the present invention is not particularly limited as long as it is a living cell or tissue, and is suitable as a method for staining cells or tissue derived from animals including humans. The type and form of the cell or tissue is not particularly limited, and may be a living cell or tissue separated from a living body, or fixed, frozen, paraffin-embedded, sectioned after being separated from a living body. It may be a cell or tissue subjected to a known pretreatment in a conventional staining method such as the above.

《Dyeing method》
A first aspect of the present invention includes a compound represented by the following formula (1), a compound represented by the formula (2), a compound represented by the formula (3), a compound represented by the formula (4), and A staining step X of staining a cell or tissue with a staining solution containing one or more compounds selected from the group of compounds represented by formula (5), and staining a substance having an aldehyde group contained in the cell or tissue. This is a dyeing method.

[In formula (1), at least one of six hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 6. , A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (2), at least one of the 10 hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 10, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (3), at least one of the eight hydrogen atoms is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 8, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (4), the four hydrogen atoms at the 3, 4, 5, 6 positions of the diaminobenzene structure may be substituted with an arbitrary substituent, and the hydrogen atoms at the 3, 4 positions are removed to form carbon. 4 to 7 cycloalkanes Q may be condensed, and one or more of the methylene groups constituting the cycloalkanes Q are (—O—) groups or (— Optionally substituted by an NH-) group;
In formula (5), at least one of the three hydrogen atoms of benzofurazan is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 3 And a hydrogen atom not substituted with R may be substituted with any substituent. ]

In the formulas (1) to (5), the hydrogen atom bonded to the ring structure may be substituted with any substituent. Examples of the optional substituent include, for example, a linear or branched aliphatic saturated hydrocarbon group having 1 to 8 carbon atoms, a linear or branched aliphatic unsaturated hydrocarbon group having 1 to 8 carbon atoms, and sulfone. Examples thereof include an acid group (—SO ₃ H), a carboxylic acid group (—COOH), an acetyl group (—COCH ₃ ), an alkoxy group having 1 to 4 carbon atoms, a halogen atom, and a hydroxyl group. Further, as the optional substituent, one or more methylene groups constituting the aliphatic saturated hydrocarbon group or the aliphatic unsaturated hydrocarbon group may be —O— except that oxygen atoms are bonded to each other. And a substituent substituted by -C (= O)-, -C (= O) O-, -OC (= O)-, or -NH-.
When the arbitrary substituent is an acid group having a negative charge, the acid group may form a salt such as a sodium salt or a potassium salt. When the arbitrary substituent has a hydrogen atom, the hydrogen atom may be substituted with a halogen atom.

In the formulas (1) to (5), the R (functional group R) that replaces the hydrogen atom bonded to the ring structure is terminated with an amino group (—NH ₂ ) or a hydrazine structure (—NH—NH ₂ ). Have.
The functional group R may contain a divalent organic group having 1 to 10 carbon atoms.
Examples of the functional group R include — (CH ₂ ) _r —NH ₂ , — (CH ₂ ) _r —NH—NH ₂ , —NH— (CH ₂ ) _r —NH ₂ , and the like. In these functional groups R, r is an integer of 0 to 10, and when one or more methylene groups are included, each methylene group is —O—, —C (, except when oxygens are bonded to each other. ═O) —, —C (═O) O—, —O—C (═O) —, —NH—, or —S— may be substituted.
When a plurality of functional groups R are present in the formulas (1) to (5), these functional groups R may be the same as or different from each other.

  As the cycloalkane Q which may be condensed to the diaminobenzene structure of the formula (4), a cycloalkane having 5 to 6 carbon atoms in which one or more methylene groups may be substituted with oxygen is preferable. A specific example of a structure having such a condensed ring is a 1,2-methylenedioxybenzene structure.

  The compounds represented by the formulas (1) to (5) (hereinafter referred to as compounds (1) to (5)) have one or more functional groups R having an amino group or a hydrazine structure at the terminal. . For this reason, a staining solution containing any one or more compounds selected from the compounds (1) to (5) is contacted with a cell or tissue to be stained and stained (hereinafter referred to as CHO staining). The functional group R of the compounds (1) to (5) can be bound to a substance having an aldehyde group (—CHO) contained in the cell or the tissue.

The substance to which the compounds (1) to (5) bind by CHO staining is a substance in which an aldehyde group is formed by pretreatment of cells or tissues before staining, even though it does not inherently have an aldehyde group. There may be. For example, when cells (gland cells) that are present in tissue sections and produce mucus containing polysaccharides are stained with CHO, the tissue sections are treated with periodic acid to oxidize the polysaccharides contained in the tissue sections. And a pretreatment for oxidizing a hydroxyl group in a polysaccharide molecule to an aldehyde group. The reaction formula explaining this pretreatment is shown below. In the following reaction formula, R ¹ and R ² represent a part of the molecular chain of the polysaccharide.

  The polysaccharide is not particularly limited as long as it is a polysaccharide that is oxidized by an oxidizing agent to generate an aldehyde group, and examples thereof include a polysaccharide having glucose as a structural unit. Specific examples include glycogen, mucus protein, glycoprotein, glycolipid and the like.

Formed after binding a substance having an aldehyde group (—CHO) contained in the cell or the tissue by CHO staining and any one of the compounds (1) to (5) by a Schiff reaction (Schiff base) For the purpose of stabilizing, a reductive amination reagent can be used. By stabilizing the Schiff base with a reductive amination reagent, the color development of the fluorescence derived from the compounds (1) to (5) can be improved.
Examples of the reductive amination reagent include 2-picoline borane. Among these, 2-picoline borane can be used regardless of the type of solvent and is preferable because it is a stable compound. The reductive amination reagent can be added in advance to a staining solution containing any one of the compounds (1) to (5) used in CHO staining at an appropriate concentration.

  Among the compounds used for CHO staining, compounds (1) to (3) and compound (5) have fluorescence in their skeleton structures themselves. Compound (4) acquires fluorescence by reacting with the aldehyde group of the substance possessed by the dyeing target to form a ring structure (see the reaction formula described below). Therefore, the compounds (1) to (5) bonded to the substance having an aldehyde group emit fluorescence after being excited by light. By observing this fluorescence with a fluorescence microscope or the like, the distribution of the substance having an aldehyde group in a CHO-stained cell or tissue can be examined.

The following reaction formula shows how an example of the compound (4) reacts with a substance having an aldehyde group to form a fluorescent ring structure. In the following reaction formula, R ³ represents an organic group constituting the substance.

  The fluorescence wavelength emitted after the compounds (1) to (5) are bonded to the substance depends largely on the skeletal structure of the compound at the time of light emission, and is excited at a wavelength in the vicinity of 365 nm to 405 nm and observed as fluorescence having a wavelength in the visible region. Is done. These fluorescence wavelengths do not overlap with the fluorescence in the visible wavelength range that occurs at the excitation wavelengths (365 nm to 405 nm) of the staining materials used in the existing hematoxylin-eosin staining (HE staining), Papanicolaou staining, and Giemsa staining. Therefore, both existing staining and CHO staining can be performed on the same staining target. This is a great advantage in the field of pathological examination. For example, by performing both HE staining and CHO staining on a tissue section, the presence or absence of a substance stained by CHO staining (for example, presence or absence of a gland cell containing a polysaccharide) is examined at a site of interest by HE staining of the tissue section. be able to. In the conventional pathological examination, HE staining is performed on the first tissue section, and when a portion to be examined is observed, a second tissue section adjacent to the first tissue section is stained with PAS staining, A method for examining the presence or absence of polysaccharides at the site to be examined is being carried out. In this conventional method, since the first tissue section and the second tissue section are strictly observed at different physical positions, the substances or cells contained in the first tissue section are the first. In reality, it can happen that it is not included in the second tissue section. For this reason, there are not a few situations in which accurate pathological diagnosis cannot be performed. Further, since two types of staining are separately performed on two tissue sections, more time and cost for staining work are required. However, as proposed by the present invention, pathological diagnosis is performed more accurately and rapidly by performing both the existing staining selected from HE staining, Papanicolaou staining, Giemsa staining, and the like and CHO staining on the same tissue section. be able to. Furthermore, since the low molecular weight compounds (1) to (5) having higher cell / tissue permeability than the giant antibody used in the conventional immunostaining method are used, the target substance is stained with higher sensitivity. can do.

The compounds (1) to (5) used in the CHO staining are excited with a wavelength in the ultraviolet region (around 365 nm to 405 nm) after or before binding to the substance, and emit fluorescence with a wavelength in the visible region. Since this fluorescence is generated by ultraviolet excitation, it can be distinguished from autofluorescence (red to green) of a staining substance used in existing staining such as HE staining caused by visible light, and is suitable for observation with a fluorescence microscope. Moreover, since it is almost colorless when observing a visible light region with a normal optical microscope, it does not disturb the existing optical microscope observation after staining such as HE staining. Therefore, CHO staining with compounds (1) to (5) is particularly useful when both existing staining and CHO staining are performed on the same staining object. Furthermore, compounds (1) to (5) can be used by mixing with a staining solution containing hematoxylin, can be used in parallel with CHO staining in parallel with conventional HE staining, and encapsulated with a curing agent after CHO staining. Since there is little fading after the treatment, the CHO-stained specimen can be stored semipermanently and has excellent advantages.
Specific examples of preferred compounds (1) to (5) are shown below.

(Dyeing process X)
As a specific method of the staining step X, for example, in a staining solution containing any one or more compounds selected from the group of compounds represented by the compounds (1) to (5) at an arbitrary concentration, cells or Examples include a method of immersing the tissue at room temperature for about 15 to 30 minutes and then removing the tissue and washing excess compound with water.
Examples of the total concentration of the one or more compounds contained in the staining liquid include 0.01 g / L to 0.2 g / L.
From the viewpoint of facilitating the formation of a Schiff base by reacting the amino group or hydrazine structure of the one or more compounds with an aldehyde group contained in the cell or tissue, the pH of the staining solution is acidic. It is preferable. In order to make the pH of the staining solution acidic, for example, hydrochloric acid is preferably added to the staining solution at a concentration of about 0.02 mol / L.
Furthermore, it is desirable to add a reductive amination reagent in order to stabilize the Schiff base. For example, 2-picoline borane may be added to a concentration of about 50 mg / L.
The staining solution may contain other optional components as long as the CHO staining is not inhibited.

(Oxidation process)
In the staining method according to the first aspect of the present invention, before the staining step X, the cells or tissues to be stained are oxidized in advance, so that at least a part of the hydroxyl groups of the polysaccharide contained in the cells or tissues is obtained. You may have the oxidation process process changed to an aldehyde group. The oxidation treatment process may be regarded as a sub-process included in the dyeing process X, or may be regarded as a process independent of the dyeing process X.

  As a method of the oxidation treatment step, for example, a known oxidation treatment method performed by conventional PAS staining for staining polysaccharides can be applied. The specific method includes, for example, a method in which a cell or tissue to be stained is immersed in a known oxidizing agent for about 15 to 30 minutes at room temperature and then lightly washed with water. Specific examples of the oxidizing agent will be described later.

(Dyeing process H)
The staining method of the first aspect of the present invention may have a staining step H for staining a basic substance contained in the cells or tissues before or after the staining step X.
Both staining step X and staining step H can be performed on the same cell or tissue sample.
The basic substance is a substance having a negative (-) charge in the cell or tissue, such as a phosphate group or a carboxyl group, and examples thereof include a cell nucleus.
As a method for staining the basic substance, the conventional hematoxylin staining (H staining) in which hematoxylin is stained as a pigment (color rack) is preferable. H staining can be performed in the same manner as in the conventional method using a staining solution containing hematoxylin.
Further, instead of H staining, the basic substance may be stained by nuclear staining using methyl green or Cologne Echlot funnel. Similarly to hematoxylin, methyl green and cologne Echlot funnel do not emit blue fluorescence and do not interfere with CHO staining.

  In the staining method of the first aspect of the present invention, the staining step X and the staining step H may be performed simultaneously. For example, CHO staining and H staining can be performed simultaneously. Examples of the method for simultaneously performing CHO staining and H staining include a method in which a mixed staining solution obtained by mixing a staining solution for CHO staining and a staining solution for H staining is brought into contact with cells or tissues and stained simultaneously. Specifically, for example, cells or tissues are immersed in a mixed staining solution containing any one or more compounds selected from the group of compounds represented by compounds (1) to (5) and hematoxylin at an arbitrary concentration. And dyeing them.

(Dyeing process E)
The staining method according to the first aspect of the present invention further includes a staining step E for staining an acidic substance contained in the cells or tissues before or after the staining step X or before or after the staining step H. Also good.
At least one of the staining step X and the staining step H and the staining step E can be performed on the same cell or tissue sample.
The acidic substance is a substance having a positive (+) charged site in the cell or tissue, and examples thereof include a cytoplasmic component.
The method for staining the acidic substance is preferably, for example, eosin staining (E staining) in which conventional eosin is used as a dye (color anion) and is dyed red by binding to a positively charged portion of the cell or tissue. E staining can be performed in the same manner as in the conventional method using a staining solution containing water-soluble eosin Y.

  Since the conventional hematoxylin-eosin staining (HE staining) performs the staining step E after the staining step H, the order in which the staining step E is performed after the staining step H is also preferable in the staining method of the first aspect of the present invention. In this order, the dyeing process X (eg CHO dyeing) can be performed before or after the dyeing process H (eg H dyeing) or after the dyeing process E (eg E dyeing). An example of the order of performing the dyeing process X, the dyeing process H, and the dyeing process E is shown in the flowchart of FIG.

(Encapsulation process)
After each of the above-mentioned staining steps, the cells or tissues can be immersed in an alcohol solution whose alcohol concentration has been increased stepwise, dehydrated, and subjected to a penetration treatment with xylol, followed by encapsulation.
When the encapsulated cell or tissue is observed with a fluorescence microscope, blue fluorescence derived from the compounds (1) to (5) bonded to the aldehyde group constituting the cell or tissue can be measured. For example, it is observed that glandular cells containing oxidized polysaccharides are stained with blue fluorescence.
Further, when the enclosed cells or tissues are observed with an optical microscope, a color derived from the dye (staining agent) used in the staining process H and the staining process E is observed. For example, cell nuclei stained blue by H staining and cytoplasm stained red by E staining are observed.

<Combination with Papanicolaou staining and Giemsa staining>
As shown in Examples described later, in the staining method of the first aspect of the present invention, conventional Papanicolaou staining or Giemsa staining can be performed on the same staining object before or after the staining step X. Papanicolaou dyeing and CHO dyeing, and Giemsa dyeing and CHO dyeing do not interfere with each other, and show the same good dyeing results as when each dyeing is carried out independently.

《Dyeing agent》
The second aspect of the present invention is represented by the compound represented by the formula (1), the compound represented by the formula (2), the compound represented by the formula (3), and the formula (4). A staining agent comprising a compound and one or more compounds (1) to (5) selected from the group of compounds represented by formula (5).
The form of the staining agent may be a solid such as a powder or a tablet containing the compounds (1) to (5), and the compounds (1) to (5) are dissolved or dispersed in any solvent at any concentration. Liquid may be used. The solvent is not particularly limited as long as it can dissolve or disperse the compounds (1) to (5). For example, purified water, primary alcohols such as methanol, ethanol, isopropanol, acetonitrile, DMSO, hexane, etc. Other organic solvents are mentioned.
The staining agent of the second aspect may further contain hematoxylin.

《Dyeing kit》
The third aspect of the present invention is a staining kit having the staining agent of the second aspect.
The staining kit may have an oxidizing agent that oxidizes a tissue section before staining in advance.
Examples of the oxidizing agent include a compound Ox that oxidizes a polysaccharide to generate an aldehyde group. Specific examples of the compound Ox include compounds such as sodium periodate. The form of the oxidizing agent may be a solid such as a powder or a tablet containing the compound Ox, or may be a liquid in which the compound Ox is dissolved or dispersed in an arbitrary solvent at an arbitrary concentration.
Prior to use of the staining kit, the staining agent of the second aspect and the oxidizing agent are preferably stored in separate containers.

The staining kit may have a stain H containing hematoxylin separately from other stains. The form of the staining agent H may be a solid such as a powder or tablet containing hematoxylin, or may be a liquid in which hematoxylin is dissolved or dispersed in an arbitrary solvent at an arbitrary concentration.
In the staining kit, hematoxylin may be included in the staining agent of the second aspect at any concentration as a component of the staining agent of the second aspect.

The staining kit may have a staining agent E containing eosin. The form of eosin contained in staining agent E may be a solid such as powder or tablet containing eosin, or may be a liquid in which eosin is dissolved or dispersed in an arbitrary solvent at an arbitrary concentration.
Prior to use of the staining kit, the staining agent and staining agent H of the second aspect, and the staining agent E are preferably stored in separate containers.

  Any other drug, container, instruction manual, and the like included in a commercially available staining kit used for staining cells or tissue sections may be included in the staining kit.

[Reagent preparation]
<CHO stain (1)>
7-Amino-4-methylcoumarin (AMC) (0.01 g) and 1 mol / L hydrochloric acid (100 μl) were dissolved in 50% ethyl alcohol (10 ml). A solution (0.5 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml) was added to the resulting solution (about 10 ml) to prepare a CHO staining agent (1).
<CHO stain (2)>
5-Amino-1-naphthalenesulfonic acid sodium salt hydrate (SAN) (0.005 g) and 1 mol / L hydrochloric acid (100 μl) were dissolved in 50% ethyl alcohol (10 ml). A solution (0.5 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml) was added to the resulting solution (about 10 ml) to prepare a CHO staining agent (2).
<CHO stain (3)>
1,2-Diamino-4,5-methylenedioxybenzene, dihydrochloride (MDB) (0.0025 g) and 1 mol / L hydrochloric acid (100 μl) were dissolved in 50% ethyl alcohol (10 ml). A solution (0.5 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml) was added to the obtained solution (about 10 ml) to prepare a CHO staining agent (3).

<CHO stain (4)>
3-amino-coumarin (100 mg / ml, DMSO solution) (10 μl) in 50% ethyl alcohol (10 ml) and a solution (1 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml). Then, RO water was added to make up to 50 ml. 1 mol / L hydrochloric acid (100 μl) was added to the resulting solution (50 ml) to prepare a CHO staining agent (4).
<CHO stain (5)>
Prepare CHO stain (5) in the same manner as CHO stain (4), except that 1-amino-pyrene (100 mg / ml, DMSO solution) (10 μl) was used instead of 3-amino-coumarin. did.
<CHO stain (6)>
CHO except that 4- (2-aminoethylamino) -7- (N, N-dimethylsulfamoyl) benzofurazan (100 mg / ml, DMSO solution) (10 μl) was used instead of 3-amino-coumarin. A CHO staining agent (6) was prepared in the same manner as the staining agent (4).
<CHO stain (7)>
In place of 3-amino-coumarin, CHO staining agent (4) except that mono-7-amino-1,3-naphthalenedisulfonic acid potassium monohydrate (100 mg / ml, aqueous solution) (10 μl) was used. In the same manner, a CHO dye (7) was prepared.
<CHO stain (8)>
CHO staining was carried out in the same manner as in the CHO staining agent (8) except that 7-amino-4- (trifluoromethyl) coumarin (100 mg / ml, aqueous solution) (10 μl) was used instead of 3-amino-coumarin. Agent (8) was prepared.

<CHO stain (9)>
4-Chloro-1,2-phenylenediamine (100 mg / ml, DMSO solution) (10 μl) was added to 50% ethyl alcohol (10 ml), and RO water was added to make up to 50 ml. 1 mol / L hydrochloric acid (100 μl) was added to the resulting solution (50 ml) to prepare a CHO staining agent (9).
<CHO stain (10)>
Similar to CHO stain (4) except that 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (100 mg / ml, aqueous solution) (10 μl) was used instead of 3-amino-coumarin. Thus, a CHO dye (10) was prepared.
<CHO stain (11)>
The same as CHO stain (4) except that 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (10 mg / ml, DMSO solution) (10 μl) was used instead of 3-amino-coumarin. Thus, a CHO dye (11) was prepared.
<CHO stain (12)>
8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (100 mg / ml, DMSO solution) (10 μl) was added to a mixture (50 ml) of ethyl alcohol: acetic acid = 9: 1 (volume ratio). Further, a solution (1 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml) was added to prepare a CHO staining agent (12).
<CHO stain (13)>
To a mixed solution (50 ml) of ethyl alcohol: acetic acid = 9: 1 (volume ratio), 8-aminopyrene-1,3,6-trisulfonsan trisodium salt (10 mg / ml, DMSO solution) (10 μl) was added. Further, a solution (1 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml) was added to prepare a CHO stain (13).
<CHO stain (14)>
To 50% ethyl alcohol (10 ml), 7-amino-4-methylcoumarin-3-acetic acid (0.007 g) was added, and RO water was added to make up to 50 ml. A solution (1 ml) of 2-picoline borane (0.05 g) dissolved in ethyl alcohol (10 ml) and 1 mol / L hydrochloric acid (100 μl) were added to the obtained solution (50 ml), and a CHO staining agent (14) Was prepared.

<Meyer's hematoxylin solution>
A solution A in which potassium aluminum sulfate (50 g) was dissolved in purified water (300 ml) was obtained.
Hematoxylin (3 g) is dissolved in purified water (600 ml), sodium iodate (0.5 g) is added and dissolved in the solution A, and the solution A is added and mixed. Chloral hydrate (50 g) and monohydrate citric acid A Japanese product (1.2 g) was further added and dissolved to obtain a Mayer's hematoxylin solution.
<Eosin solution>
Eosin Y (1 g) was dissolved in distilled water (100 ml) and precipitated by adding 10 ml of 1N hydrochloric acid. The obtained precipitate was washed with purified water and air-dried to obtain powder Y. Next, powder Y (0.5 g) was added to ethyl alcohol (1000 ml) to obtain a storage solution Y. At the time of use, solution Y (60 ml) and acetic acid (0.01 ml) were added to purified water (140 ml) to obtain an eosin solution.
<Gill's Hematoxylin Solution>
Ethylene glycol (250 ml), hematoxylin (2 g), sodium iodate (0.2 g), aluminum sulfate (17.6 g) and acetic acid (20 ml) were dissolved in purified water (730 ml) to obtain a hematoxylin solution of gill. .

[Example 1; CHO staining]
First, a paraffin-embedded section of human tissue prepared by a conventional method was used to obtain a tissue section that had been deparaffinized by a known method of immersing in the order of xylene, alcohol, and ion-exchanged water.
Next, the tissue section was immersed in a 0.5% aqueous sodium periodate solution for 20 minutes at room temperature, and then washed with water. By this oxidation treatment, at least a part of the hydroxyl groups of the polysaccharide in the tissue slice was changed to aldehyde groups.
Subsequently, the tissue section was immersed in 50 ml of CHO staining agent (1) for 20 minutes at room temperature, and then washed with water. By this staining, a tissue section was obtained that was subjected to CHO staining in which AMC binds to the polysaccharide in which the aldehyde group was formed.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method to obtain a tissue section sample that was stained with CHO.

An image obtained by observing the tissue section specimen with a fluorescence microscope is shown in FIG.
FIG. 2 is a fluorescence image obtained by observing a blue wavelength region with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.

[Example 2; CHO staining]
CHO staining was performed in the same manner as in Example 1 except that the CHO staining agent (2) was used.
The image observed with the fluorescence microscope of the obtained tissue slice sample is shown in FIG.
FIG. 3 is a fluorescence image obtained by observing a blue wavelength region with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.

[Example 3; CHO staining]
CHO staining was performed in the same manner as in Example 1 except that the CHO staining agent (3) was used.
The image observed with the fluorescence microscope of the obtained tissue slice sample is shown in FIG.
FIG. 4 is a fluorescence image obtained by observing the blue wavelength region with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.

[Example 4; H staining + CHO staining → E staining]
First, a paraffin-embedded section of human tissue prepared by a conventional method was used to obtain a tissue section that had been deparaffinized by a known method of immersing in the order of xylene, alcohol, and ion-exchanged water.
Next, the tissue section was immersed in a 0.5% aqueous sodium periodate solution for 20 minutes at room temperature, and then washed with water. By this oxidation treatment, at least a part of the hydroxyl groups of the polysaccharide in the tissue slice was changed to aldehyde groups.
Subsequently, the tissue section was immersed in a solution prepared by mixing 10 ml of the CHO stain (1) with 40 ml of the Meyer's hematoxylin solution at room temperature for 20 minutes, and then washed with water. By this staining, a tissue section was obtained in which CHO staining in which AMC binds to the polysaccharide having aldehyde groups formed and hematoxylin staining in which hematoxylin binds to basic substances such as cell nuclei was obtained. .
Next, the tissue section was immersed in the eosin solution for 3 minutes at room temperature, and then washed with water. By this staining, a tissue section was obtained that was stained with eosin that binds eosin to acidic substances such as cytoplasm.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method, and a tissue section specimen subjected to CHO staining and HE staining was obtained.

Images obtained by observing the tissue section specimen with an optical microscope and a fluorescence microscope are shown in FIGS.
FIG. 5 is a transmission image observed with a normal optical microscope. Tissue structures such as cell nuclei (blue) and cytoplasm (red to pink) can be clearly identified.
FIG. 6 is a fluorescence image obtained by observing the blue wavelength region with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly distinguished on a black background.
FIG. 7 is a fluorescence image obtained by observing the green wavelength region with a fluorescence microscope. Autofluorescence of eosin has been observed. 5 to 7, it was confirmed that the CHO staining and the HE staining did not interfere with each other, and each staining was sufficiently performed, and that the images were high-quality images that could be used for pathological diagnosis.

[Example 5: H staining + CHO staining]
The tissue section oxidized in the same manner as in Example 4 was immersed in a solution obtained by mixing 10 ml of the CHO staining agent (1) and 40 ml of the above-mentioned gil hematoxylin solution. Then, it lightly washed with water and immersed in 0.5% ethyl alcohol to wash excess hematoxylin. Otherwise, as in Example 4, the same tissue section was simultaneously subjected to CHO staining and Gill's hematoxylin staining.

  As a result, as in Example 4, a clear nuclear structure stained in blue by Gill's hematoxylin staining was observed in the transmission image of the optical microscope (FIG. 8). Furthermore, tissue structures such as glandular cells containing polysaccharides were clearly observed without interference by Gill's hematoxylin staining in the fluorescence image of the fluorescence microscope (FIG. 9).

[Example 6; CHO staining → H staining → E staining]
First, similarly to Example 4, a tissue section subjected to deparaffinization and oxidation treatment was obtained.
Next, the tissue section was immersed in a solution obtained by mixing 10 ml of CHO staining agent (1) in 40 ml of distilled water at room temperature for 20 minutes, and then washed with water. By this staining, a tissue section was obtained that was subjected to CHO staining in which AMC binds to the polysaccharide in which the aldehyde group was formed.
Subsequently, the tissue section was immersed in the Mayer's hematoxylin solution for 3 minutes at room temperature, and then washed with water. By this staining, a tissue section stained with hematoxylin that binds hematoxylin to basic substances such as cell nuclei was obtained.
Finally, eosin staining was performed in the same manner as in Example 4 to obtain a tissue section sample.

  In this example, the same tissue section was stained with CHO and then with HE. As a result, as in Example 4, a clear tissue structure stained in blue and red by HE staining was observed in the transmission image of the optical microscope (FIG. 10). Furthermore, tissue structures such as glandular cells containing polysaccharides were clearly observed without interference by HE staining in the fluorescence image of the fluorescence microscope (FIG. 11).

[Example 7; H staining → E staining → CHO staining]
First, in the same manner as in Example 1, deparaffinized tissue sections were obtained. This tissue section was subjected to a staining step without being oxidized.
Next, the tissue section was immersed in the Mayer's hematoxylin solution for 5 minutes at room temperature, and then washed with water. By this staining, a tissue section stained with hematoxylin that binds hematoxylin to basic substances such as cell nuclei was obtained.
Subsequently, the tissue section was immersed in the eosin solution for 5 minutes at room temperature, and then washed with water. By this staining, a tissue section was obtained that was stained with eosin that binds eosin to acidic substances such as cytoplasm.
Next, the tissue section was immersed in a solution in which 10 ml of CHO staining agent (1) was mixed with 40 ml of distilled water for 15 minutes at room temperature and then washed with water. By this staining, a tissue section was obtained that was subjected to CHO staining in which AMC binds to the polysaccharide in which the aldehyde group was formed.
Finally, dehydration and encapsulation were performed in the same manner as in Example 4 to obtain a tissue section sample.

  In this example, the same tissue section was stained with HE and then stained with CHO. As a result, as in Example 4, a clear tissue structure stained in blue and red by HE staining was observed in the transmission image of the optical microscope (FIG. 12). Furthermore, tissue structures such as glandular cells containing polysaccharides were clearly observed without interference by HE staining in the fluorescence image of the fluorescence microscope (FIG. 13).

[Example 8; CHO staining → Papanicolaou staining]
Similar to the cell sample subjected to conventional Papanicolaou staining, a cell sample subjected to deparaffinization treatment was obtained. This cell specimen is fixed on a glass slide so that it can be handled in the same manner as a tissue section.
First, the cell sample was oxidized in the same manner as in Example 4.
Subsequently, the cell specimen was immersed in a solution obtained by mixing 10 ml of the CHO staining agent (1) and 40 ml of the gil hematoxylin solution at room temperature for 20 minutes and then washed with water. Further, after fractionation with 0.5% hydrochloric acid alcohol, the ethanol concentration was increased stepwise from 70% ethanol to 95% ethanol for affinity.
Subsequently, the obtained cell specimen was sequentially stained by a conventional method using a commercially available orange G-6 stain (OG-6) and a commercially available EA50 stain.
Finally, it was dehydrated with ethanol and sealed with xylene to obtain a cell specimen.

  In this example, the same cell specimen was subjected to CHO staining and Papanicolaou staining. As a result, a clear tissue structure stained in blue to red similar to normal Papanicolaou staining was observed in the transmission image of the optical microscope (FIG. 14). Furthermore, tissue structures such as glandular cells containing polysaccharides were clearly observed without interference by Papanicolaou staining in the fluorescence image of the fluorescence microscope (FIG. 15).

[Example 9: CHO staining → Giemsa staining]
In the same manner as the blood cell sample subjected to conventional Giemsa staining, the blood cell sample was applied to a slide glass and fixed with 100% methanol.
First, the blood cell sample was oxidized in the same manner as in Example 4.
Subsequently, the blood cell specimen was immersed in 50 ml of CHO staining agent (1) at room temperature for 20 minutes and then washed with water.
Subsequently, the obtained blood cell specimen was stained by a conventional method using a commercially available Giemsa staining solution. After lightly washing with water, the sample was dried, immersed in xylene and sealed to obtain a blood cell specimen.

  In this example, the same blood cell specimen was subjected to CHO staining and Giemsa staining. As a result, in the transmission image of the optical microscope, it was stained blue-purple similarly to normal Giemsa staining (FIG. 16). Furthermore, blue fluorescence was observed at a site where polysaccharides were present without interference by Giemsa staining in the fluorescence image of the fluorescence microscope (FIG. 17).

[Example 10; CHO staining]
First, a paraffin-embedded section of human tissue prepared by a conventional method was used to obtain a tissue section that had been deparaffinized by a known method of immersing in the order of xylene, alcohol, and ion-exchanged water.
Next, the tissue section was immersed in a 0.5% aqueous sodium periodate solution for 20 minutes at room temperature, and then washed with water. By this oxidation treatment, at least a part of the hydroxyl groups of the polysaccharide in the tissue slice was changed to aldehyde groups.
Subsequently, the tissue section was immersed in 50 ml of CHO staining agent (4) for 20 minutes at room temperature, and then washed with water. By this staining, a tissue section was obtained that was subjected to CHO staining in which 3-aminocoumarin was bound to the polysaccharide on which an aldehyde group was formed.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method to obtain a tissue section sample that was stained with CHO.

Images obtained by observing the tissue section specimen with a fluorescence microscope are shown in FIGS.
FIG. 18 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 19 is a fluorescence image obtained by observing a green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 11; CHO staining]
CHO staining was performed in the same manner as in Example 10 except that the CHO staining agent (5) was used.
Images of the obtained tissue slice specimens observed with a fluorescence microscope are shown in FIGS.
FIG. 20 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 21 is a fluorescence image obtained by observing a green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 12; CHO staining]
CHO staining was performed in the same manner as in Example 10 except that the CHO staining agent (6) was used.
Images of the obtained tissue slice specimens observed with a fluorescence microscope are shown in FIGS.
FIG. 22 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of the glandular cells (blue green) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 23 is a fluorescence image obtained by observing the green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 13; CHO staining]
CHO staining was performed in the same manner as in Example 10 except that the CHO staining agent (7) was used.
Images of the obtained tissue section specimen observed with a fluorescence microscope are shown in FIGS.
FIG. 24 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 25 is a fluorescence image obtained by observing a green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 14; CHO staining]
CHO staining was performed in the same manner as in Example 10 except that the CHO staining agent (8) was used.
Images of the obtained tissue section specimen observed with a fluorescence microscope are shown in FIGS.
FIG. 26 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of the glandular cells (blue green) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 27 is a fluorescence image obtained by observing the green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 15; CHO staining]
A tissue section subjected to deparaffinization treatment and oxidation treatment in the same manner as in Example 10 was obtained.
Next, the tissue section was immersed in 50 ml of CHO staining agent (9) heated to 55 ° C. for 15 minutes, and then washed with water. By this staining, a tissue section was obtained that was subjected to CHO staining in which 4-chloro-o-phenylenediamine was bonded to the polysaccharide in which the aldehyde group was formed.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method to obtain a tissue section sample that was stained with CHO.

Images obtained by observing the tissue section specimen with a fluorescence microscope are shown in FIGS.
FIG. 28 is a fluorescence image obtained by observing the blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of the glandular cells (blue green) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 29 is a fluorescence image obtained by observing the green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 16; CHO staining]
A tissue section subjected to deparaffinization treatment and oxidation treatment in the same manner as in Example 10 was obtained.
Next, the tissue section was immersed in 50 ml of CHO staining agent (10) heated to 55 ° C. for 30 minutes, and then washed with 2 mM sodium hydroxide aqueous solution. By this staining, a tissue section was obtained in which CHO staining was performed in which 8-aminonaphthalene-1,3,6-trisulfonic acid was bound to the polysaccharide in which the aldehyde group was formed.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method to obtain a tissue section sample that was stained with CHO.

Images obtained by observing the tissue section sample with a fluorescence microscope are shown in FIGS.
FIG. 30 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 31 is a fluorescence image obtained by observing the green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 17; CHO staining]
CHO staining was performed in the same manner as in Example 16 except that the CHO staining agent (11) was used.
Images obtained by observing the obtained tissue section specimen with a fluorescence microscope are shown in FIGS. 32 and 33. FIG.
FIG. 32 is a fluorescence image obtained by observing a blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.
FIG. 33 is a fluorescence image obtained by observing the green wavelength region (480 nm) with a fluorescence microscope. The organizational structure can be clearly identified.

[Example 18; CHO staining]
First, a paraffin-embedded section of human tissue prepared by a conventional method was used to obtain a tissue section that had been deparaffinized by a known method of immersing in the order of xylene, alcohol, and ion-exchanged water.
Next, the tissue section was immersed in a 0.5% aqueous sodium periodate solution for 20 minutes at room temperature, and then washed with water. By this oxidation treatment, at least a part of the hydroxyl groups of the polysaccharide in the tissue slice was changed to aldehyde groups.
Subsequently, the tissue section was immersed in 50 ml of CHO staining agent (14) for 20 minutes at room temperature, and then washed with water. By this staining, a tissue section was obtained that was subjected to CHO staining in which 7-amino-4-methylcoumarin-3-acetic acid was bound to the polysaccharide in which the aldehyde group was formed.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method to obtain a tissue section sample that was stained with CHO.

FIG. 34 shows an image obtained by observing the tissue section specimen with a fluorescence microscope.
FIG. 34 is a fluorescence image obtained by observing the blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.

[Example 19: H staining + CHO staining → E staining]
The tissue section oxidized in the same manner as in Example 18 was immersed in a solution obtained by mixing 20 ml of the CHO staining agent (14) and 20 ml of the Meyer's hematoxylin solution. Then, it washed lightly.
Subsequently, the tissue section washed with water in the eosin solution was immersed for 3 minutes at room temperature and then washed with water. By this staining, a tissue section was obtained that was stained with eosin that binds eosin to acidic substances such as cytoplasm.
Finally, dehydration treatment and encapsulation treatment were performed on the tissue section by a conventional method, and a tissue section specimen subjected to CHO staining and HE staining was obtained.

A transmission image obtained by observing the tissue section sample with a microscope is shown in FIG. 35, and an image observed with a fluorescence microscope is shown in FIG.
FIG. 36 is a fluorescence image obtained by observing the blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.

[Example 20; H staining + CHO staining]
The tissue section oxidized in the same manner as in Example 18 was immersed in a solution obtained by mixing 20 ml of the CHO staining agent (14) and 20 ml of the Meyer's hematoxylin solution. Then, it washed lightly.
Thereafter, the tissue slice was subjected to dehydration treatment and encapsulation treatment by a conventional method to obtain a tissue slice specimen subjected to CHO staining and H staining.

FIG. 37 shows a transmission image obtained by observing the tissue section sample with a microscope, and FIG. 38 shows an image observed with a fluorescence microscope.
FIG. 38 is a fluorescence image obtained by observing the blue wavelength region (365 nm) with a fluorescence microscope. The tissue structure of glandular cells (blue) containing polysaccharides can be clearly identified on a black background, and it was confirmed that the images were of high quality that could be used for pathological diagnosis.

[Comparative Example 1]
According to the procedure of Example 4, except that fluorescein isothiocyanate (FITC) (0.005 g) having a skeletal structure not corresponding to compounds (1) to (5) was used instead of compound (1). Staining was performed on the same tissue section.
As a result, in the transmission image of the optical microscope, yellow coloration by FITC interfered with blue and red color by HE staining, and it was difficult to observe the tissue structure in the same manner as conventional HE staining.
On the other hand, it was predicted that the fluorescence wavelength (yellow) by FITC could be observed in the fluorescence image of the fluorescence microscope, but when actually performed, the fluorescence could not be observed. This is probably because eosin staining was performed under acidic conditions, so that fluorescence was not generated from FITC or quenched.
From the above results, the staining result by FITC could not be fully grasped.

[Comparison experiment]
First, as in Example 1, two tissue sections subjected to deparaffinization were obtained. These tissue sections are cut from sites adjacent to each other in the tissue.
The obtained first tissue section was stained with PAS by a conventional method. The second tissue section was stained with CHO using the CHO stain (1) in the same manner as in Example 1. After staining, dehydration and encapsulation were performed by a conventional method to obtain two tissue section samples.
Both the transmission image (FIG. 39) obtained by observing the PAS-stained specimen with an optical microscope and the fluorescence image (FIG. 40) obtained by observing the CHO-stained specimen with a fluorescence microscope were clear images.
Comparing both images, CHO staining had higher resolution (resolution) between the stained sites and superior sensitivity than PAS staining.

  The configurations and combinations thereof in the embodiments described above are examples, and additions, omissions, substitutions, and other modifications of known configurations are possible without departing from the spirit of the present invention.

  The present invention can be widely applied to technical fields for staining cells and tissue sections, for example, medical and medical fields such as pathological diagnosis and cytohistological research.

Claims (16)

A substance having an aldehyde group contained in the cells or tissues by staining the cells or tissues with a staining solution containing one or more compounds selected from the group of compounds represented by the following formulas (1) to (5) A dyeing method comprising a dyeing step X.
[In formula (1), at least one of six hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 6. , A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (2), at least one of the 10 hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 10, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (3), at least one of the eight hydrogen atoms is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 8, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (4), the four hydrogen atoms at the 3, 4, 5, 6 positions of the diaminobenzene structure may be substituted with an arbitrary substituent, and the hydrogen atoms at the 3, 4 positions are removed to form carbon. The cycloalkane Q of formula 4-7 may be condensed, and one or more methylene groups constituting the cycloalkane Q are a (—O—) group or (— Optionally substituted by an NH-) group;
In formula (5), at least one of the three hydrogen atoms of benzofurazan is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 3 And a hydrogen atom not substituted with R may be substituted with any substituent. ]   The staining method according to claim 1, further comprising an oxidation treatment step in which at least a part of hydroxyl groups of the polysaccharide contained in the cells or tissues is changed to aldehyde groups by oxidizing the cells or tissues in advance.   The staining method according to claim 1 or 2, further comprising a staining step H for staining a basic substance contained in the cell or tissue before or after the staining step X.   The staining method according to claim 3, wherein in the staining step H, the basic substance is stained using a staining solution containing hematoxylin. For the cells or tissues, the staining step X;
The staining method according to claim 1 or 2, wherein the staining step H for staining a basic substance contained in the cell or tissue is simultaneously performed.   The dyeing | staining method of Claim 5 using the dyeing | staining liquid containing 1 or more types of compounds chosen from the group of the compound represented by said Formula (1)-(5), and hematoxylin.   The dyeing | staining process E which dye | stains the acidic substance contained in the said cell or tissue with respect to the said cell or tissue which passed through the said dyeing | staining process X and the said dyeing | staining process H is further included in any one of Claims 3-6. Dyeing method.   The staining method according to claim 7, wherein in the staining step E, the acidic substance is stained with a staining solution containing eosin.   The staining method according to claim 1, comprising the staining step X and a HE staining step for the same tissue section.   The staining method according to claim 1, comprising the staining step X and a step of performing Papanicolaou staining on the same cell specimen.   The staining method according to claim 1, comprising the staining step X and Giemsa staining on the same blood specimen. A staining agent comprising one or more compounds selected from the group of compounds represented by the following formulas (1) to (5).
[In formula (1), at least one of six hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 6. , A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (2), at least one of the 10 hydrogen atoms is substituted with R, R is an amino group or a functional group having a hydrazine structure at the end, and n is an integer of 1 to 10, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (3), at least one of the eight hydrogen atoms is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 8, A hydrogen atom not substituted with R may be substituted with any substituent;
In formula (4), the four hydrogen atoms at the 3, 4, 5, 6 positions of the diaminobenzene structure may be substituted with an arbitrary substituent, and the hydrogen atoms at the 3, 4 positions are removed to form carbon. The cycloalkane Q of formula 4-7 may be condensed, and one or more methylene groups constituting the cycloalkane Q are a (—O—) group or (— Optionally substituted by an NH-) group;
In formula (5), at least one of the three hydrogen atoms of benzofurazan is substituted with R, R is a functional group having an amino group or a hydrazine structure at the end, and n is an integer of 1 to 3 And a hydrogen atom not substituted with R may be substituted with any substituent. ]   The staining agent according to claim 12, further comprising hematoxylin.   A staining kit comprising the staining agent according to claim 12 or 13.   The staining kit according to claim 14, comprising the staining agent and a staining agent containing hematoxylin.   The staining kit according to claim 14 or 15, further comprising a staining agent containing eosin.