WO2017164355A1 - Method for producing particles for pathological staining use, particles for pathological staining use, and washing method - Google Patents

Abstract

A method for producing particles for pathological staining use, comprising polycondensing a compound represented by general formula (1) to produce the particles. In formula (1), R¹'s independently represent a hydrogen atom, CH₂OR² or CH₂OH, and R² represents an organic group, wherein at least one of R¹'s represents CH₂OH.

Description

Method for producing pathological staining particle, pathological staining particle and washing method

The present invention relates to a method for producing pathological staining particles, a particle for pathological staining, and a washing method, and more specifically, a method for producing particles that are used for pathological staining and have good reproducibility of bright spot sensitivity and number of bright spots. The present invention relates to a treatment for washing pathological staining particles and staining slides produced by the method using an acidic aqueous solution.

Conventionally, pathological diagnosis is performed as one of medical diagnosis. A pathologist diagnoses a disease from data indicating the result of a biopsy performed on a tissue piece collected from a human body by pathological diagnosis, and informs a clinician whether treatment or surgery is necessary. Depending on the patient's condition and pathological diagnosis, the medical doctor decides the drug treatment policy, and the surgical doctor decides whether or not to perform the operation.

In order to provide data for determining whether or not the subject is suffering from the target disease, immunostaining is performed on the tissue sections and the like of the subject. In this immunostaining, for example, an antigen is fluorescently labeled by specifically binding a fluorescently labeled antibody to an in vivo molecule (antigen) whose expression level is increased or decreased depending on the presence or absence of the disease, and the amount of fluorescent signal is used to determine the disease. Quantifying the amount of antigen associated with. Thereby, data for diagnosing whether or not the subject suffers from the target disease is provided. As a conventional immunostaining method, a method (enzyme antibody method) using an antibody labeled with an enzyme that generates a dye when a predetermined substrate is added has been employed. In recent years, an immunostaining method (fluorescent antibody method) using an antibody labeled with a fluorescent substance having better discrimination has been used. As a technique for binding fluorescently labeled antibodies to antigens, a modified scaffold is created by modifying the surface of a particle for pathological staining in which a fluorescent dye is encapsulated in the particle or fixed on the particle surface, and the antibody is directly or indirectly bound to this modified scaffold. There is known a technique for binding to an antigen and binding it to an antigen. In this technique, in order to obtain effective data, it is required that the sensitivity of the bright spot observed based on the fluorescent label and the reproducibility of the number of bright spots are good.

It is known that the sensitivity of the bright spot and the reproducibility of the bright spot number depend on the modified scaffold containing an amino group on the particle surface. However, since it is difficult to increase the amount of this modified scaffold in the conventional pathological staining technology, the sensitivity of the bright spot and the reproducibility of the number of bright spots, especially the sensitivity of the bright spot at low expression and the reproducibility of the number of bright spots are improved. It was difficult to do.

As a method for surface modification of particles, Patent Document 1 discloses that nanoparticles and a ligand interaction agent are associated with each other, the ligand interaction agent is crosslinked with a crosslinking agent such as hexamethoxymethylmelamine, and then the surface modification is performed on the ligand interaction agent. A method of binding a ligand is disclosed. However, even if the nanoparticle contains a fluorescent dye in this method and is used as a particle for pathological staining, the amount of the modified scaffold cannot be increased, and the improvement of the sensitivity of the bright spot and the reproducibility of the number of bright spots is not possible. It was difficult.

In order to provide the data for the diagnosis, a tissue section (tissue specimen) is prepared by slicing a tissue specimen obtained by organectomy or needle biopsy to a thickness of several microns, and the tissue section is subjected to a predetermined staining process. In order to obtain various findings after observation, observation using an optical microscope or a fluorescence microscope is widely performed. Here, when using particles containing fluorescent dyes as described above for fluorescent labeling, unlike conventional immunostaining methods using an enzyme, the dyes do not mix. Thus, in addition to staining by immunostaining, staining for cell morphology observation can be performed simultaneously. In many cases, tissue sections are prepared by dehydrating and fixing paraffin blocks to fix the collected tissues, then slicing them to a thickness of several μm and removing the paraffin. Here, since the tissue slice hardly absorbs and scatters light and is almost colorless and transparent, morphological observation staining for observing the cell morphology of the tissue slice, for example, two dyes of hematoxylin and eosin is used prior to the above observation. Hematoxylin and eosin staining (HE staining) is standard. Examples of other morphological observation staining include Papanicolaou staining (Pap staining) used in cytodiagnosis, for example.

For example, when hematoxylin and eosin staining (HE staining), which is standard for observing the morphology of cells or tissues, is used, the cell nucleus, lime, cartilage tissue, bacteria, and mucus are colored blue-blue to light by hematoxylin staining. It is stained blue, and cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are stained red to dark red by eosin staining. The tissue sections thus immunostained are sealed with a mounting medium containing an anti-fading agent, and then a fluorescence image is taken while irradiating with a predetermined excitation light. (Since eosin emits fluorescence, the eosin stained image can also be taken as a fluorescence image). Then, the captured fluorescent image labeled with the antigen protein and the (fluorescence) image for cell morphology observation that can specify the position of the cell membrane are superimposed, and the bright spots observed in the cell membrane region per cell are superimposed. The number is measured, and whether or not the antigen protein is abnormally expressed is determined by the value.

In the embodiment in which the morphological observation staining process is performed simultaneously with the immunostaining method (fluorescent antibody method) as described above, it is preferable to use fluorescent dye-containing nanoparticles as a phosphor. Patent Document 2 (International Publication WO2013 / 035703 pamphlet), Patent Document 3 (International Publication WO2013 / 147081 pamphlet) and the like can be referred to.

JP-T-2015-511167 International Publication WO2013 / 035703 Pamphlet International Publication WO2013 / 147081 Pamphlet

The present invention relates to a method for producing particles for pathological staining with good sensitivity of bright spots and reproducibility of the number of bright spots, in particular, particles for pathological staining with good sensitivity and reproducibility of the number of bright spots at low expression ( Hereinafter, it is referred to as “the manufacturing method of the present invention”). A second object of the present invention is to provide particles for pathological staining.

When the pathological staining particles used in the tissue evaluation method by the conventional immunostaining method are used in combination with a staining solution (for example, a hematoxylin solution and / or an eosin solution which is a staining solution for morphological observation), There is a problem that the luminance for pathological staining is lowered. In addition, the luminance reduction rate of the pathological staining particles varies depending on the staining treatment with the staining solution and is not constant, so that the variation in the luminance of the stained pathological staining particles becomes large (that is, cannot be corrected uniformly). There was also a problem.

That is, the present invention performs a process (fluorescent labeling process) for labeling a target biological substance contained in a tissue section on a specimen slide with a particle for pathological staining by an immunostaining method, and subsequently the tissue section subjected to the fluorescent labeling process. It is a third object to provide a method capable of suppressing a decrease in luminance and variation in luminance of a stained slide even when a treatment (staining treatment) is performed for staining the specimen with a staining liquid for morphological observation.

In one aspect, the present invention for achieving the above object is as follows:
Provided is a method for producing particles for pathological staining in which particles are produced by condensation polymerization of a compound represented by the following general formula (1).

(In Formula (1), R ¹ is each independently a hydrogen atom, CH ₂ OR ² or CH ₂ OH, R ² is an organic group, and at least one of R ¹ is CH ₂ OH. )
In another aspect, the present invention provides particles for pathological staining having particles containing a polycondensation product of a compound represented by the following general formula (1).

(In Formula (1), R ¹ is each independently a hydrogen atom, CH ₂ OR ² or CH ₂ OH, R ² is an organic group, and at least one of R ¹ is CH ₂ OH. )
In a further aspect of the present invention, a process of fluorescently labeling a target biological substance contained in a tissue section on a specimen slide with the aforementioned pathological staining particles (fluorescence labeling process) by immunostaining.
A method for washing a stained slide in which the fluorescently labeled tissue section is stained with a staining solution for morphology observation (staining process), and the stained tissue section is washed with an acidic aqueous solution (washing process). provide.

According to the method for producing pathological staining particles of the present invention, the sensitivity of the bright spot and the reproducibility of the number of bright spots observed based on the fluorescent label, in particular, the sensitivity of the bright spot at low expression and the reproducibility of the number of bright spots are good. Certain particles for pathological staining can be produced.

The particles for pathological staining of the present invention are excellent in the sensitivity of the bright spot and the reproducibility of the number of bright spots observed based on the fluorescent label, and particularly reproduce the sensitivity of the bright spot and the number of bright spots at low expression. As described above, according to the present invention, it is possible to provide particles for pathological staining exhibiting excellent diagnostic ability capable of well reproducing the sensitivity of bright spots and the number of bright spots at low expression.

In addition, the staining slide prepared using the pathological staining particles of the present invention is washed with an acidic aqueous solution, so that the specimen slide can be stained with a morphological observation staining solution such as hematoxylin. Variations in luminance can be reduced and luminance reduction can be suppressed.

This makes it possible to acquire a fluorescent image with improved signal accuracy corresponding to the target protein, and more accurately evaluate the expression level of the target protein, leading to improved reliability of pathological diagnosis.

It is a flowchart which shows the example of embodiment in the case of dyeing | staining based on the immuno-staining method of the washing | cleaning method of the dyeing | staining slide produced using the particle | grains for pathological staining produced with the manufacturing method of this invention.

The present invention is a method for producing particles for pathological staining in which particles are produced by condensation polymerization of a compound represented by the following general formula (1) (hereinafter also referred to as compound (1)). Moreover, the particle | grains for pathological staining of this invention have the particle | grains containing the polycondensation product of a compound (1).

In the formula (1), six R ^{1 s} are each independently a hydrogen atom, CH ₂ OR ² or CH ₂ OH, and R ² is an organic group. The organic group is not particularly limited as long as the compound (1) can undergo polycondensation, and examples thereof include an alkyl group, and specific examples include a methyl group and an ethyl group.

Of the six R ¹ , at least one is CH ₂ OH, preferably at least two are CH ₂ OH. That is, the compound represented by the formula (1) preferably has 1 to 6 CH ₂ OH groups in one molecule and has 2 to 6 CH ₂ OH groups. In the six R ^{1 s} , the total of CH ₂ OH and CH ₂ OR ² is preferably 4 to 6, more preferably 5 to 6, and particularly preferably 6.

If two of R ¹ is a CH ₂ OH, which R ¹ of the six R ¹ in Formula (1) is no problem even CH ₂ OH. Two R ¹ possessed by one amino group out of the three amino groups contained in the formula (1) may be both CH ₂ OH, and one of the two R ¹ possessed by one amino group is CH ₂ OH, and one of the two R ¹ possessed by the other one amino group may be CH ₂ OH. Even if three of R ¹ is a CH ₂ OH, which R ¹ of the six R ¹ is not may be a CH ₂ OH, formula (1) has three amino groups contained in the two having each one of R ¹ is may be CH ₂ OH, are two of R ¹ are both CH ₂ OH having one amino group, one of the two R ¹ with the other one amino group May be CH ₂ OH. Even if four of R ¹ is a CH ₂ OH, which R ¹ of the six R ¹ is not may be a CH ₂ OH, formula (1) has two amino groups contained in the two having each may R ¹ is a both CH ₂ OH, are two of R ¹ are both CH ₂ OH having one amino group, one is CH of the two R ¹ with the other two amino groups, each _It may be ₂ OH.

As for the hydrogen atom and CH ₂ OR ² represented by R ^{1 in} formula (1), the number and bonding position thereof are not limited as long as at least one of R ¹ is CH ₂ OH.

In the compound represented by the formula (1), examples of the compound in which ^one of R ¹ is CH ₂ OH include compounds represented by the following formula (2) or (3), and three of R ¹ are CH ₂ OH. As an example of the compound which is, the compound represented by following formula (4) is mentioned.

In the production method of the present invention, compound (1) may be used alone or in combination of two or more compounds (1) represented by different chemical formulas. That is, a mixture of two or more compounds selected from the compound (1) may be used. A mixture of compounds represented by formula (1) having different numbers of CH ₂ OH groups, wherein the average number of CH ₂ OH groups contained in one molecule is 1 to 6. Also good.

Conventionally, particles for pathological staining produced from melamine resin have been produced by polycondensing a compound represented by the following formula (5).

The compound represented by the following formula (5) (hereinafter also referred to as compound (5)) has an OCH ₃ group but does not have an OH group, and therefore, melamine resin particles obtained by polycondensing compound (5). Also has an OCH ₃ group but no OH group. When an antibody is bound to melamine resin particles, generally, a bifunctional amine is reacted with melamine resin particles, and the particle surface is modified with a group containing an amino group to form a modified scaffold, and the antibody is directly or directly attached to the modified scaffold. Join indirectly. Since the bifunctional amine does not easily react with the OCH ₃ group, a large amount of the modified scaffold cannot be introduced into the melamine resin particles obtained by condensation polymerization of the compound (5). For this reason, there is a limit to the amount of antibody that can be bound to conventional pathological staining particles prepared from melamine resin. As a result, conventional pathological staining particles prepared from a melamine resin containing a fluorescent dye did not have good sensitivity and reproducibility of the number of bright spots observed based on the fluorescent label.

On the other hand, the method for producing particles for pathological staining of the present invention produces particles by condensation polymerization of compound (1). Compound (1) has at least one CH ₂ OH group as described above. For this reason, the melamine resin particles obtained by polycondensation of the compound (1) necessarily have OH groups based on CH ₂ OH groups that were not involved in the polycondensation. Since the OH group easily reacts with the bifunctional amine, it is easy to react the bifunctional amine with the melamine resin particles obtained by condensation polymerization of the compound (1) to produce a modified scaffold. It is possible to introduce a large amount of modified scaffolds. Therefore, many antibodies can be bound to the pathological staining particles obtained by polycondensation of the compound (1), and the density of the fluorescent dye in the pathological staining particles can be increased. As a result, the pathological dyeing particles produced from the melamine resin containing the fluorescent dye obtained by the production method of the present invention have good sensitivity and reproducibility of the number of bright spots observed based on the fluorescent label. Become.

As can be seen from the above description, in order to introduce a large amount of the modified scaffold, the melamine resin particles preferably have many OH groups. The more the compound (1) has more CH ₂ OH groups, the more OH groups based on the CH ₂ OH groups that did not participate in the condensation polymerization remain. Therefore, in the production method of the present invention, the compound (1) It is more preferable to have many CH ₂ OH groups. Thus, six of R ¹ of formula (1), the one is always CH ₂ OH, preferably more two are CH ₂ OH, and most preferably all six is CH ₂ OH.

Compound (1) is methylolated with formaldehyde melamine, yet its reaction with methylol compound and R compound represented by ² OH, methylolated, and the extent of the reaction between the compound represented by the methylol compound and R ² OH It can be synthesized by adjusting. If methylolation is strongly performed, the number of CH ₂ OH groups introduced into the product can be increased, and if methylolation is weakened, the number of CH ₂ OH groups introduced into the product can be decreased. it can. Further, if the reaction between the methylolated compound and the compound represented by R ² OH is not carried out or if it is weakened, the number of remaining CH ₂ OH groups can be increased, and the reaction with the compound represented by R ² OH If it is carried out strongly, the number of remaining CH ₂ OH groups can be reduced.

The melamine resin used in the method for producing a particle for pathological staining of the present invention includes, for example, R ¹ which is CH ₂ OR ² among compounds (1), and the compound in which R ² is an alkyl group includes melamine and formaldehyde Can be synthesized by synthesizing methylol melamine by the reaction with, and further etherifying the methylol melamine with an alcohol represented by R ² OH.

These reactions can be performed according to a conventional method.

Reaction conditions for polycondensation of the compound represented by the formula (1) may be in accordance with conventionally known synthesis conditions for melamine resins.

The phosphor-integrated nanoparticles are nano-sized (with a diameter of less than 1 μm) that are integrated by encapsulating a plurality of phosphors, such as the fluorescent dyes described later, in a base material or attaching them to the surface. It is a particulate phosphor. In the production method of the present invention, the compound (1) can be polycondensed in the presence of a fluorescent dye. When polycondensation is performed in this manner, the compound (1) undergoes polycondensation while wrapping the fluorescent dye, so that melamine resin particles containing the fluorescent dye can be obtained. Moreover, since the melamine resin has a fine network structure, the encapsulated fluorescent dye is unlikely to leak from the melamine resin particles.

Another embodiment of the production method of the present invention includes a method for producing particles for pathological staining in which a fluorescent dye is added to and contained in particles obtained by condensation polymerization of compound (1). By adding and containing a fluorescent dye after such condensation polymerization, it is possible to adsorb the fluorescent dye on the surface of the melamine resin or to enclose the fluorescent dye inside the melamine resin. By such a method, the melamine resin particle | grains which have a fluorescent pigment | dye which are particles for pathological dyeing | staining can be obtained.

Therefore, in the present invention, the phosphor-integrated nanoparticles refer to particulate phosphors that are integrated by encapsulating a fluorescent dye in a melamine resin or attaching it to the surface. The use of such phosphor-integrated nanoparticles in immunostaining is equivalent to the use of a single fluorescent molecule labeled with a target biomolecule as compared with the case where the fluorescent substance is used alone (a single fluorescent dye molecule). It is preferable because the intensity of the fluorescence emitted from the light can be increased, noise such as autofluorescence of the cells and distinguishability from other dyes can be enhanced, and fading caused by irradiation with excitation light can be suppressed.

The fluorescent dye is not particularly limited, but is preferably an organic fluorescent dye from the viewpoint of immunostaining, such as rhodamine dyes, BODIPY (registered trademark, manufactured by Invitrogen), squarylium dyes, aromatic dyes, and the like. Can be mentioned.

Among these, aromatic dyes such as aromatic hydrocarbon dyes, rhodamine dyes and the like are preferable because of their relatively high light resistance, and among them, perylene, pyrene, and perylene belonging to aromatic dyes. Diimide is preferred. Furthermore, rhodamine dyes and perylene diimide are excellent in quantum yield, light absorption, and the like, and are excellent in luminous efficiency. Therefore, resin particles containing these have excellent emission intensity compared to resin particles containing other dyes.

Specific examples of rhodamine dyes include 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, Texas Red, Spectrum Red, LD700 PERCHLORATE, those Derivatives and the like.

Specific examples of the BODIPY dye include BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, 3061 BODIPY 650/665 (manufactured by Invitrogen), derivatives thereof and the like.

Specific examples of the squarylium-based dye include SRfluor680-Carboxylate, 1,3-Bis [4- (dimethylamino) -2-hydroxyphenyl] -2,4-dihydroxycyclobutenedidium dihydroxide, bis, 1,3-bis (1,3-Bis (1,3-Bis) phenyl] -2,4-dihydroxycyclobutenedidium dihydroxide, bis, 2- (4- (Diethylamino) -2-hydroxyphenyl) -4- (4- (diethyliminothio) -2-hydroxy-cyclodioxy-3cyclo-cyclodioxy-3cyclo-cyclodioxy-3cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo-cyclo) 1-enolat , 2- (4- (Dibutylamino) -2-hydroxyphenyl) -4- (4- (dibutylimino) -2-hydroxycyclohexa-2,5-diylenedene) -3-oxoxyclobut-1-enolate, 2- (8-Hydroxy- 1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido [3,2,1-ij] quinolin-9-yl) -4- (8-hydroxy-1,1, 7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyrido [3,2,1-ij] quinolinium-9 (5H) -ylidene) -3-oxocyclobut-1-enolate, induction thereof And the like.

Specific examples of the aromatic hydrocarbon dye include N, N-Bis- (2,6-diisopropylphenyl) -1,6,7,12- (4-tert-butylphenoxy) -perylene-3,4,9, 10-tetracarbonacidimide, N, N′-Bis (2,6-diisopropyphenyl) -1,6,7,12-tetraphenoxyperylene-3, 4: 9,10-tetracarbodiimide, N, N′-bis (2,6-pylido (2,6-pylidine) ) Perylene-3,4,9,10-bis (dicarbimide), 16, N, N'-Bis (2,6-dimethylphenyl) perylene-3,4,9,10-tetracarb xylicimide, 4,4 ′-[(8,16-Dihydro-8,16-dioxodibenzo [a, j] perylene-2,10-diyl) dioxy] dibutylic acid, 2,10-Dihydroxy-dibenzo [a, j] perylene-8,16-dione, 2,10-Bis (3-aminopropoxy) dibenzo [a, j] perylene-8,16-dione, 3,3 '-[(8,16-Dihydro-8,16-dioxodibenzo [a, j] perylene-2,10-diyl) dioxy] dipropylamine, 17-BIS (Octyloxy) Anthra [9,1,2-cde-] Benzo [RST] Pentaphene-5-10-Dione, Octade canoicacid, 5,10-dihydro-5,10-dioxanthanth [9,1,2-cde] benzo [rst] pentaphene-16,17-diylester, Dihydroxydibenzanthrone, Benzensulfonicacid, 4,4 ', 4' '-[[2,9-bis [2,6-bis (1-methylethyl) phenyl] -1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxanthra [2,1 , 9-def: 6,5,10-d'e'f '] diisoquinoline-5,6,12,13-tetrayl] tetrakis (oxy)] tetrakis-, Benzenethanamine, 4,4', 4 '', 4 '' '-[[2,9-bis [2, 6-bis (1-methylethyl) phenyl] -1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra [2,1,9-def: 6,5,10-d 'e'f'] diisoquinoline-5,6,12,13-tetrayl] tetrakis (oxy)] tetrakis [N, N, N-trimethyl-], and derivatives thereof.

The amount of the fluorescent dye added is usually 17 to 27 mg, preferably 20 to 25 mg, relative to 1 g of compound (1).

As described above, the average particle size of the pathological staining particles produced by polycondensation of the compound (1) is usually 10 to 500 nm, preferably 50 to 200 nm. The variation coefficient of the average particle diameter is preferably 5 to 20%, more preferably 5 to 15%. The average particle size of the particles and the coefficient of variation thereof shown in the present invention are obtained by performing SEM observation of the particles, arbitrarily measuring the particle size of 1000 particles from the obtained SEM image, and calculating the average particle from the measured values. The diameter and its coefficient of variation.

In the production method of the present invention, as described above, the melamine resin particles obtained by polycondensation of the compound represented by the formula (1) are reacted with a bifunctional amine to form an amino group serving as a modified scaffold on the surface of the particles. A group containing can be introduced.

The bifunctional amine is not particularly limited as long as a modified scaffold capable of directly or indirectly binding an antibody can be introduced. From the viewpoint of efficient antibody binding, methanediamine, polyethylene glycol diamine, etc. Is preferred. Moreover, it can replace with the said bifunctional amine and can also use another amination reagent. Examples of other amination reagents include aminopropyltrimethoxysilane and aminopropyltriethoxysilane.

The reaction of the bifunctional amine with the melamine resin particles may be performed according to the conventionally known reaction conditions of the bifunctional amine with the melamine resin particles.

Particles having a group containing an amino group serving as a modified scaffold on the surface obtained by reacting a melamine resin particle obtained by polycondensation of compound (1) with a bifunctional amine are prepared according to a conventional method. The antibody can be directly or indirectly bound to the. Thus, the particle | grains which couple | bonded the antibody can be used for an immuno-staining according to a conventional method. As described above, when immunostaining is performed using the pathological staining particles obtained by polycondensation of the compound (1), the sensitivity of the bright spots observed based on the fluorescent label and the reproducibility of the number of bright spots are good. Is obtained. In particular, even with low expression where satisfactory data could not be obtained with conventional pathological staining particles, good sensitivity of the bright spot and reproducibility of the number of bright spots can be obtained.

Further, the particles for pathological staining of the present invention can be obtained by the above production method. The particle for pathological staining of the present invention can suppress the variation in the luminance of the fluorescent dye and the decrease in luminance by the staining slide cleaning method of the present invention, which will be described later. It is unclear whether it is brought about. Although it can be presumed that the structure of the surface of the particle for pathological staining has an influence, it is difficult to clearly show the necessary structure in order to exhibit the effect obtained by the method for washing a staining slide of the present invention.

The particles for pathological staining produced by the method for producing particles for pathological staining of the present invention are used for, for example, a process (fluorescent labeling process) for labeling a target biological substance contained in a tissue section on a specimen slide by immunostaining. Subsequently, a staining slide is produced by performing a process (staining process) for staining the tissue section that has been subjected to the fluorescent labeling process with a staining solution for morphology observation.

The pathological staining particles (hereinafter also referred to as staining particles or simply referred to as particles) produced by the method for producing pathological staining particles of the present invention are used, for example, in a staining slide by performing the following treatment. be able to.

That is, a process of labeling a target biological substance contained in a tissue section on a specimen slide with the pathological staining particles of the present invention (fluorescence labeling process), and a process of staining the fluorescently labeled tissue section with a staining solution for morphology observation (Staining process) Furthermore, the dyeing | staining slide is produced by the process (washing | cleaning process) which wash | cleans the tissue section dye | stained.

Here, when washing the stained tissue section, it is preferable to use an acidic aqueous solution as the washing solution. By washing with an acidic aqueous solution, when the specimen slide is stained with a staining solution such as hematoxylin or hematoxylin-eosin, the variation in luminance of the particles for pathological staining is further reduced, and further reduction in luminance can be suppressed. To. As a result, a fluorescent image with improved signal accuracy corresponding to the target protein can be obtained, and the expression level of the target protein can be more accurately evaluated, leading to an increase in the reliability of pathological diagnosis.

Thus, when the specimen slide is stained by the procedure as described above, the reason why the above effect can be obtained by washing the specimen slide with an acidic aqueous solution is not clear, but is presumed as follows. That is, it is speculated that the staining liquid for morphological observation such as hematoxylin and eosin is adsorbed to the particles for pathological staining, resulting in a decrease in brightness and variations, and the pathological staining particles of the morphological observation staining liquid by washing with an acidic aqueous solution. It is considered that the adsorption to the surface is suppressed, the brightness of the particles for pathological staining is kept relatively high, and variation is also suppressed.

The acidic aqueous solution used in the washing treatment performed when preparing a staining slide using the pathological staining particles prepared by the method for producing pathological staining particles of the present invention has a pH of 2 or more and less than 7. Preferably, the pH is 3 or more and 6.5 or less.
(Preparation method of stained slide)
Hereinafter, a method for producing a stained slide will be described.

The entire process for producing the staining slide can be mainly classified into “specimen pretreatment process”, “staining process”, and “specimen posttreatment process”.

The above-mentioned “specimen pretreatment step” generally includes deparaffinization treatment, antigen activation treatment, washing treatment and the like.

In the “staining step”, a treatment for fluorescent labeling based on an immunostaining method (immunostaining treatment), that is, a primary antibody treatment depending on whether the target biological substance is directly labeled or indirectly labeled Secondary antibody processing, fluorescent labeling processing, morphology observation staining processing, and the like are included.

The above “specimen post-treatment process” includes a washing process, an encapsulation process, and a solvent replacement process and a dehydration process as necessary.

Hereinafter, processing necessary for carrying out the present invention will be further described. Items not specifically described in the present specification, for example, general items and processes required to complete a slide stained based on an immunostaining method, and observation using the completed stained slide The photographing process, the image processing / analysis process using the photographed image, and the like can be appropriate according to the descriptions in Patent Documents 2 to 3 and other general technical matters.

Hereinafter, the “fluorescence labeling process” (immune staining process) and the “morphological observation staining process” included in the “staining process” will be described.

<Fluorescent labeling treatment (immunostaining treatment)>
The fluorescence labeling process included in the “staining step” is a process of labeling a target protein with a pathological staining particle based on an immunostaining method.

When performing the labeling process based on the immunostaining method, the specimen slide that has undergone the activation process is immersed in the labeling solution for immunostaining, and one or more labeling agents in the labeling solution are targeted directly or indirectly. Label by binding to protein (antigen).

The target protein (antigen) is not particularly limited, but typically, it can be a target gene for pathological diagnosis based on immunostaining, such as HER2, TOP2A, HER3, EGFR, P53, MET, and other various types. Select from proteins derived from cancer / tumor-related genes (so-called biomarker genes), as well as cancer-related proteins such as cancer growth factors, transcription regulatory factors, growth regulatory factor receptors, and transcription regulatory factor receptors. be able to. Therefore, the antibody described below can also be prepared based on a known technique as having the binding ability suitable for the target protein (antigen) as described above, and can also be obtained as a commercial product.

There are various methods for immunostaining, and it is not particularly limited as long as it can stain a tissue section so that the target protein can be fluorescently labeled and used for pathological diagnosis etc. Some examples include:
A method of preparing a fluorescently labeled primary antibody in which a phosphor and a primary antibody are linked, and directly fluorescently labeling and staining the target protein with the fluorescently labeled primary antibody (primary antibody method);
Prepare a primary antibody and a fluorescently labeled secondary antibody in which a fluorescent label and a secondary antibody are linked, react the primary antibody with the target protein, and then react the fluorescently labeled secondary antibody with the primary antibody Indirectly fluorescently labeling and staining the target protein (secondary antibody method)
Prepare a biotin-modified primary antibody in which a primary antibody and biotin are linked, and an avidin-modified phosphor in which a phosphor and avidin or streptavidin are linked. After reacting the target protein with a biotin-modified primary antibody, further avidin modification A method in which a fluorescent substance is reacted and the target protein is indirectly fluorescently labeled and stained using the avidin-biotin reaction (primary antibody method using avidin-biotin in combination);
Prepare a primary antibody, a biotin-modified secondary antibody in which a secondary antibody and biotin are linked, and an avidin-modified phosphor in which a fluorophore and avidin or streptavidin are linked, react the primary antibody with the target protein, and then modify the biotin A method in which a secondary antibody is reacted, and then an avidin-modified phosphor is further reacted, and the target protein is indirectly fluorescently labeled and stained using the avidin-biotin reaction (second antibody method combined with avidin-biotin).

In the above-described primary antibody method using avidin-biotin combination or secondary antibody method using avidin-biotin, a hapten is used instead of biotin and avidin. Low molecular weight substances) and anti-hapten antibodies, such as dicoxygenin and anti-dicoxygenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antigens, and other substances with similar specific reactivity may also be utilized. it can.

The immunostaining treatment may be performed according to standard procedures and processing conditions for each of the various methods described above. In general, a specimen slide on which a specimen is placed may be immersed in one or more reagents according to the immunostaining method under an appropriate temperature and time condition (for example, overnight at 4 ° C.). . Various reagents necessary for immunostaining, that is, fluorescently labeled primary / secondary antibodies, biotin-modified primary / secondary antibodies, avidin-modified secondary antibodies / phosphors, etc. are dissolved, and blocking agents such as BSA as necessary A solution such as a buffer solution to which is added can be prepared according to a known method, and can also be obtained as a commercial product.

After the treatment with the labeling solution, the specimen slide is preferably immersed and washed in a washing solution such as PBS. Usually, the temperature of the washing treatment using PBS performed after the treatment with the labeling solution is room temperature, and the time is 3 to 30 minutes. If necessary, PBS may be exchanged during immersion.

<Dyeing treatment for morphology observation>
Next, the “morphological observation staining process” included in the “staining process” will be described. In the “staining process for morphological observation”, after the specimen slide is labeled with a pathological staining particle as a labeling substance in the “staining step” above, the specimen slide is obtained in order to obtain cell or tissue shape and positional information of each part of the cell. It is dyed with a staining liquid for morphology observation.

Examples of the staining liquid for morphological observation include hematoxylin staining liquid, eosin staining liquid, and Papanicolaou (Pap) staining liquid.

As hematoxylin staining solution,
Mayer's hematoxylin solution (composition example: hematoxylin 1.0 g / potassium alum 50 g / sodium iodate 0.2 g / chloral hydrate 50 g / citric acid 1.0 g / distilled water 1,000 ml), Mayer hematoxylin solution (x2) (composition example: hematoxylin 2.0g / potassium alum 50g / sodium iodate 0.4g / chlorate hydrate 50g / citric acid 1.0g / distilled water 1,000ml), caratach hematoxylin solution (composition example: hematoxylin 1.0g / potassium alum 50g / sodium iodate 0.2) g / glycerin 200 ml / distilled water 800 ml), gil hematoxylin solution (No. 1, composition example: hematoxylin 2.0 g / aluminum sulfate 14-18 water 17.6 g / sodium iodate 0.2 g / ethylene glycol 250 ml / glacial acetic acid 20 ml / distilled 730 ml of water) and Lily Meyer's hematoxylin solution (example composition: 5.0 g of hematoxylin / 50 g of ammonium alum) Sodium periodate 0.5 g / glycerin 300 ml / glacial acetic acid 20 ml / distilled water 700 ml) and the like.

As eosin stain,
1% eosin Y solution (composition example: eosin Y 5.0 g / distilled water 500 ml / acetic acid few drops), 0.1% eosin Y ethanol solution (composition example: 10% eosin Y solution 5 ml / 95% ethanol 495 ml), 0. 5% eosin Y ethanol solution (composition example: 10% eosin Y solution 25ml / 95% ethanol 475ml), eosin alcohol solution, acid extract (acid extract eosin solution 100ml / 95% ethanol 800ml / acetic acid 8ml) .

The staining process using the morphology observation staining solution may be performed according to a general procedure. For example, in the case of staining with hematoxylin and eosin (HE), treatment is performed by staining with Mayer's hematoxylin solution for 5 minutes, washing with running water at 45 ° C. for 3 minutes, and then staining with 1% eosin solution for 5 minutes.

<Cleaning process>
The cleaning process performed in the “sample post-treatment process” is a process performed before the solvent replacement process (or before the dehydration process when the dehydration process is an optional process before the solvent replacement process). In this process, the stained specimen slide is washed with a predetermined acidic aqueous solution.

After performing a morphological observation staining treatment using a morphological observation staining solution such as hematoxylin or eosin described above, when performing such a cleaning treatment, remove and wash the staining solution bound to the pathological staining particles. Is considered possible. That is, the staining liquid binds not only to the tissue but also to the pathological staining particles, and deactivates the fluorescence. In addition, since the degree of binding between the staining solution and the pathological staining particles varies depending on the type of the pathological staining particles, it is considered that the luminance varies. It is considered that the washing treatment with a predetermined acidic aqueous solution weakens the bond between the pathological staining particles and the staining liquid, suppresses the deactivation of fluorescence, and enables observation of the pathological staining particles.

On the other hand, when a neutral to alkaline aqueous solution is used, the staining liquid remains bound to the pathological staining particles, and it is estimated that the luminance decreases.

In the preparation process of the specimen slide, the specimen slide may be washed after the specimen slide is treated with various solutions. The washing treatment using an acidic aqueous solution here uses a staining liquid for morphology observation. This refers to what is performed after the dyeing process for morphological observation and before the solvent replacement process (if the dehydration process is an optional step before the solvent replacement process).

(Acidic aqueous solution)
The acidic aqueous solution used for the “cleaning treatment” can be prepared by mixing acid and water in an appropriate ratio. The pH range of the acidic aqueous solution is preferably 2 or more and less than 7 and more preferably pH 3 or more and 6.5 or less in consideration of the effect of suppressing the decrease in brightness and variation. By setting the pH of the acidic aqueous solution to preferably 2 or more and less than 7, more preferably pH 3 to 6.5, the effect of suppressing variation in luminance of the particles for pathological staining is sufficiently maintained, and the effect of reducing the decrease in luminance Is expressed.

As long as the acid is well mixed with water (has compatibility), it can be used regardless of whether it is an organic acid or an inorganic acid. Examples of the organic acid include formic acid, acetic acid, citric acid, and oxalic acid. Examples of the inorganic acid include hydrochloric acid, nitric acid, phosphoric acid, and sulfuric acid. In particular, hydrochloric acid, citric acid and the like are preferably used.

The method of washing treatment using an acidic aqueous solution is not particularly limited, but in general, the staining slide may be immersed in an acidic aqueous solution contained in a container. The immersion in the acidic aqueous solution is usually performed at room temperature, and the immersion time is usually 1 second to 30 minutes, preferably 5 seconds to 15 minutes. Operations such as immersion may be repeated a plurality of times.

[Production Example 1]
Melamine and formaldehyde were mixed at a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to this mixture so as to have a pH of 9 to 14, and a heating reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reactant and methanol are mixed in a molar ratio of reactant 1 and methanol 1, sulfuric acid is added to the mixture so as to have a pH of 0 to 3, and a heating reaction is performed at 85 ° C. for 30 minutes to obtain a melamine resin. Obtained.
[Production Example 2]
Melamine and formaldehyde were mixed at a molar ratio of melamine 1 and formaldehyde 5, sodium hydroxide was added to the mixture so as to have a pH of 9 to 14, and a heating reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reactant and methanol are mixed in a molar ratio of reactant 1 and methanol 1, sulfuric acid is added to the mixture so as to have a pH of 0 to 3, and a heating reaction is performed at 85 ° C. for 30 minutes to obtain a melamine resin. Obtained.

[Production Example 3]
Melamine and formaldehyde were mixed in a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to the mixture so as to have a pH of 9 to 14, and a reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reactant and methanol are mixed in a molar ratio of reactant 1 and methanol 2, and sulfuric acid is added to the mixture so as to have a pH of 0 to 3, followed by a heating reaction at 85 ° C. for 30 minutes to obtain a melamine resin. Obtained.
[Production Example 4]
Melamine and formaldehyde were mixed at a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to this mixture so as to have a pH of 9 to 14, and a heating reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reactant and methanol are mixed in a molar ratio of reactant 1 and methanol 3, and sulfuric acid is added to the mixture so as to have a pH of 0 to 3, followed by heating at 85 ° C. for 30 minutes, and melamine resin. Got.

[Production Example 5]
Melamine and formaldehyde were mixed at a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to this mixture so as to have a pH of 9 to 14, and a heating reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reaction product and methanol are mixed in a molar ratio of reaction product 1 and methanol 4, and sulfuric acid is added to this mixed solution so that the pH becomes 0 to 3, and a heating reaction is performed at 85 ° C. for 30 minutes to obtain a melamine resin. Obtained.
[Production Example 6]
Melamine and formaldehyde were mixed at a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to this mixture so as to have a pH of 9 to 14, and a heating reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reactant and methanol are mixed in a molar ratio of reactant 1 and methanol 5, and sulfuric acid is added to the mixture so as to have a pH of 0 to 3, followed by a heating reaction at 85 ° C. for 30 minutes to obtain a melamine resin. Obtained.

[Production Example 7]
Melamine and formaldehyde were mixed in a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to the mixture so as to have a pH of 9 to 14, and a heating reaction was performed at 70 ° C. for 30 minutes. Thereafter, the reaction product and methanol are mixed in a molar ratio of reaction product 1 and methanol 6, and sulfuric acid is added to the mixture so that the pH becomes 0 to 3, and a heating reaction is performed at 85 ° C. for 30 minutes to obtain a melamine resin. Obtained.
[Production Example 8]
Melamine and formaldehyde were mixed in a molar ratio of melamine 1 and formaldehyde 6, sodium hydroxide was added to the mixture so as to have a pH of 9 to 14, and a reaction was performed at 70 ° C. for 30 minutes to obtain a melamine resin.

[Production Example 9]
Melamine and formaldehyde were mixed in a molar ratio of melamine 1 and formaldehyde 4, sodium hydroxide was added to this mixture so as to have a pH of 9 to 14, and a reaction was performed at 70 ° C. for 30 minutes to obtain a melamine resin.

The numbers of OH groups and methoxy groups of the compounds used in the production of the melamine resins of Production Examples 1 to 9 were measured by H-NMR, thermal analysis and elemental analysis. The results are shown in Table 1.
(Sensitivity and reproducibility evaluation of bright spots of particles for pathological staining)
[Example 1]
[Manufacture of particles for pathological staining]
PI dye-containing polymelamine particles were produced as fluorescent particles as follows.

N, N′-Bis (2,6-diisopropylphenyl) -1,6,7,12-tetraphenylperylene-3,4: 9,10-tetracarboxdiimide was treated with concentrated sulfuric acid to produce a perylene diimide sulfonic acid derivative. This was converted to an acid chloride to obtain a perylene diimide sulfonic acid chloride derivative.

After adding 14.4 mg of perylene diimidesulfonic acid chloride derivative to 22.5 mL of water, the mixture was heated on a hot stirrer at 70 ° C. for 20 minutes, and 0.65 g of the melamine resin prepared in Preparation Example 1 was added to this solution. The mixture was further stirred with heating for 5 minutes. 100 μL of formic acid was added to this dispersion, heated and stirred at 60 ° C. for 20 minutes, and then allowed to cool to room temperature. After cooling, the reaction mixture was placed in a centrifuge tube and centrifuged at 12,000 rpm for 20 minutes, and the supernatant was removed. The obtained PI pigment-containing polymelamine particles were washed with ethanol and water.

[Binding of antibodies to particles for pathological staining]
0.1 mg of the obtained PI dye-containing polymelamine particles were dispersed in 1.5 mL of ethanol, and 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added to this dispersion and reacted for 8 hours. A surface amination treatment was performed.

A mixed solution of EDTA containing 2 mM of EDTA (ethylenediaminetetraacetic acid) and PBS (phosphate buffered saline) was added to the particles subjected to surface amination treatment to adjust the particle concentration to 3 nM. This dispersion was mixed with SM (PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) so as to have a final concentration of 10 mM, and reacted for 1 hour. The mixture is centrifuged at 10,000 G for 20 minutes, and the supernatant is removed. Then, a mixed solution of EDTA and PBS containing 2 mM of EDTA is added to the precipitate, the precipitate is dispersed, and centrifuged again. went. By performing washing by the same procedure three times, PI dye-containing polymelamine particles (fluorescent particles) having a maleimide group at the terminal were obtained.

On the other hand, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) is subjected to thiol group addition treatment using N-succinimidyl S-acetylthioacetate (SATA), followed by filtration through a gel filtration column to bind to PI dye-containing polymelamine particles A possible streptavidin solution was obtained.

The above-mentioned PI dye-containing polymelamine particles having a maleimide group and streptavidin were mixed in a mixed solution of EDTA containing 2 mM of EDTA and PBS, and reacted for 1 hour. 10 mM mercaptoethanol was added to the reaction solution to stop the reaction. After the obtained solution was concentrated with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification to obtain streptavidin-binding PI dye-containing polymelamine particles (pathological staining particles).
[Immunostaining (Immunohistochemistry (IHC) method)]
As a pathological section, one whose total protein amount was previously measured by ELISA was used, HER2 was used as the antigen, and cultured cells of SK-BR-3 and MDA-MB-231 were used for the determination.

Thereafter, the pathological section was immunostained using streptavidin-conjugated PI dye-containing polymelamine particles (labeled body) prepared using the melamine resin produced in Production Example 1, and then morphological observation staining (H staining) was performed. Carried out. Immunostaining and morphological observation staining were performed as follows.

The cultured cells were deparaffinized with xylene and washed with water. The washed cultured cell slide was autoclaved in a 10 mM citrate buffer (pH 6.0) at 121 ° C. for 5 minutes to activate the antigen. The cultured cell slide after the activation treatment was washed with PBS buffer, and then subjected to blocking treatment with 1% BSA-containing PBS buffer for 1 hour in a wet box. After blocking treatment, anti-HER2 rabbit monoclonal antibody (4B5) (manufactured by Ventana) diluted to 0.05 nM with 1% BSA-containing PBS buffer was reacted with the sections for 2 hours. This was washed with PBS buffer and then reacted with a biotin-labeled anti-rabbit monoclonal antibody capable of binding to 4B5 diluted to 2 μg / mL with 1% BSA-containing PBS buffer for 30 minutes. After this reaction, the aforementioned streptavidin-conjugated PI dye-containing polymelamine particles diluted to 0.2 nM with 1% BSA-containing PBS buffer were cut into sections, neutral pH environment (pH 6.9 to 7.4), room temperature The reaction was carried out for 3 hours under the conditions. Then, the cultured cell slide which performed the above-mentioned process was wash | cleaned using PBS buffer solution (pH 7.0).

After performing the above immunostaining, morphological observation staining (H staining) was performed. The immunostained cultured cell slide was stained with Mayer's hematoxylin solution for 5 minutes, and hematoxylin staining (H staining) was performed to obtain a stained slide. The sections were then washed with running water at 45 ° C. for 3 minutes.

After staining with hematoxylin, an operation of immersing in pure ethanol for 5 minutes was performed 4 times, followed by washing and dehydration. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, the tissue section was encapsulated using an encapsulant (“Enterlan New”, manufactured by Merck) to prepare a sample slide for observation.
[Observation process]
The number of bright spots of SK-BR-3 and MDA-MB-231 after staining with hematoxylin was counted, and the SN ratio was shown in Table 1 as (number of bright spots of SK-BR-3 / number of bright spots of MDA-MB231). When the SN ratio was less than 15, it was judged as x, when it was 15 or more and less than 20, it was judged as ◯, and when it was 20 or more, it was judged as ◎.

The tissue sections subjected to immunostaining and morphological observation staining were irradiated with predetermined excitation light to emit fluorescence. The tissue section in this state was observed with a fluorescence microscope (BX-53, manufactured by Olympus) and imaged. The bright spot was measured by the ImageJ FindMaxima method (exposure time was 400 ms and NoiseTolerance was 60). The wavelength of the excitation light was set to 575 to 600 nm with respect to the PI dye-containing polymelamine particles by passing through an optical filter. The range of the wavelength (nm) of fluorescence to be observed was also set to 612 to 682 nm by passing through an optical filter. The excitation wavelength condition for microscopic observation and image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm ² . The exposure time at the time of image acquisition was arbitrarily set (for example, set to 400 milliseconds) so as not to saturate the brightness of the image.
[Examples 2 to 7]
Examples 2 to 7 were carried out in the same manner as in Example 1 except that the melamine resins produced in Production Examples 2 to 7 were used in place of the melamine resin produced in Production Example 1.

The SN ratio is shown in Table 1. The determination was performed in the same manner as in Example 1.
[Comparative Examples 1 and 2]
Comparative Examples 1 and 2 were carried out in the same manner as in Example 1 except that the melamine resins produced in Production Examples 8 and 9 were used in place of the melamine resin produced in Production Example 1.

Table 1 shows the SN ratio. The determination was performed in the same manner as in Example 1.

(Examples 8 to 11: Sensitivity and reproducibility of bright spots when the particles produced in Production Examples 1 to 4 were used and washed after morphological observation staining with a hematoxylin staining solution)
The above-described H staining was carried out in the same manner as in Examples 1 to 4, and staining slides were obtained.
After the H staining treatment, each staining slide was washed according to the following procedures (i) to (ii).
(I) A pH 7 cleaning solution was prepared. Pure water was used as the cleaning liquid having a pH of 7.
(Ii) Each staining slide subjected to the staining process for morphology observation was immersed in a cleaning solution at room temperature for 4 minutes to perform a cleaning process.

(Dehydration treatment)
Each stained slide subjected to the washing treatment was immersed in dehydrated ethanol for 5 minutes. This operation was repeated 4 times to perform dehydration treatment.

(Solvent replacement process)
Each dyed slide subjected to the dehydration treatment was immersed in xylene at room temperature for 2 to 10 seconds to perform a solvent replacement treatment. This operation was repeated 4 times.

(Encapsulation processing)
The staining slide was encapsulated by the following procedures (i) to (ii).
(I) After the dyeing slide subjected to the solvent replacement treatment was dropped with Enteran New (Merck) at room temperature, it was covered with a cover glass and air-dried at room temperature for 10 minutes to perform an encapsulation treatment.
(Ii) Thereafter, the stained slide subjected to the encapsulation treatment was stored in the dark until the signal was measured.

<Fluorescence image capture and analysis>
Fluorescence was emitted by irradiating a predetermined excitation light to each stained slide after the encapsulation treatment. The stained slide in this state was observed and imaged with the aforementioned fluorescence microscope (OLYMPUS "BX-53") and the aforementioned microscope digital camera (OLYMPUS "DP73"). The excitation light was set to 575 to 600 nm by passing through an optical filter. The range of the wavelength (nm) of fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter. The conditions of the excitation wavelength during microscopic observation and image acquisition were such that the irradiation energy near the center of the field of view was 900 W / cm ² for excitation at 580 nm. The exposure time at the time of image acquisition was arbitrarily set so as not to saturate the brightness of the image (for example, set to 4000 μsec) and imaged. The number of bright spots of the HER2 (3+) tissue was an average value of 1000 cells measured by the ImageJ FindMaxims method based on an image captured at 400 times. In addition, a pathological staining particle per cell was calculated from one captured image, and a coefficient of variation (CV) was calculated as an index representing variation.
(Comparative Example 3; Sensitivity and reproducibility of bright spots when the particles produced in Production Example 9 are used and washed after morphological observation staining with a hematoxylin staining solution)
The same operation as in Example 8 was performed except that a slide sample prepared under the same conditions as those of the slide prepared in Comparative Example 2 was used.
(Examples 12 to 17: Sensitivity and reproducibility of bright spots when washed with acidic aqueous solutions having different pH after morphological observation staining with hematoxylin staining solution)
The same procedure as in Example 4 was performed until the aforementioned H staining, and a staining slide was obtained. After the H staining treatment, each staining slide was performed in the same manner as in Example 8 except that the washing treatment was changed to the following procedures (i) to (ii). (I) Six types of cleaning liquids having a pH of 1, 2, 3, 4, 5, or 6 were prepared. A washing solution having a pH of 1 to 6 was prepared by diluting 1M hydrochloric acid.
(Ii) Each staining slide subjected to the staining process for morphology observation was immersed in each cleaning solution at room temperature for 4 minutes to perform the cleaning process.

Table 2 shows the results of Examples 8 to 11 and Comparative Example 3. The stained slides of Examples 8 to 11 gave good results with a judgment of ◯ or higher. In particular, the stained slide of Example 11 using the particles produced in Production Example 4 had a large number of bright spots (S / N ratio) and small variation in luminance (CV). Then, about the Example mentioned later, it implemented by producing the dyeing | staining slide using the particle | grains produced in manufacture example 4. FIG.

The results of Examples 12 to 17 are shown in Table 2. When an acidic cleaning solution with a pH of 1 to 6 is used, the number of pathological staining particles observed is larger than when a neutral cleaning solution with a pH of 7 is used, and the variation in luminance of the pathological staining particles It can be seen that (CV) is small.

* In the table represents the PID score of a stained slide prepared using SK-BR-3 cultured cells. The PID score is an average value of analysis results obtained by counting the number of bright spots of 10 stained slides prepared using SK-BR-3 cultured cells. In the evaluation items in Table 2, “−” indicates that the evaluation item is not implemented.

(Examples 18-27: Sensitivity and reproducibility of bright spots when washed with acidic aqueous solution after morphological observation staining with hematoxylin-eosin staining solution)
(1) Specimen pretreatment step (1-1) Deparaffinization As a specimen slide of a HER2-positive stained control specimen (Cosmo Bio CB-A712 series), deparaffinization is performed according to the following procedures (i) to (iii). Processed.
(I) Immerse the specimen slide in a container containing xylene at room temperature for 30 minutes. The xylene was changed three times during the process. (Ii) The specimen slide is immersed in a container containing ethanol at room temperature for 30 minutes. The ethanol was changed three times during the process. (Iii) The specimen slide was immersed in a container containing water for 30 minutes. The water was changed three times along the way.

(1-2) Activation Treatment After the specimen slide was deparaffinized, activation treatment was performed according to the following procedures (i) to (v).
(I) The specimen slide was washed with water. (Ii) The specimen slide was immersed in a 10 mM citrate buffer (pH 6.0) for 30 minutes. (Iii) The autoclave process was performed at 121 degreeC for 10 minutes. (Iv) The specimen slide after the autoclave treatment was immersed in a container containing PBS for 30 minutes and washed. (V) 1% BSA-containing PBS was placed on the specimen slide and subjected to blocking treatment for 1 hour.

(2) Immunostaining step (2-1) Primary antibody treatment After the specimen slide was activated, the primary antibody treatment was performed. Using PBS containing 1% BSA, “anti-HER2 rabbit monoclonal antibody (4B5)” manufactured by Ventana was prepared at 0.05 nM, and the solution of the primary antibody was applied to the above-mentioned blocking-treated specimen slide. The reaction was allowed to proceed overnight at 4 ° C.

(2-2) Secondary antibody treatment The specimen slide treated with the primary antibody was washed with PBS, and then diluted to 6 μg / mL with PBS containing 1% BSA, and the biotin-modified secondary antibody solution and 30 at room temperature. Reacted for 1 minute.

(2-3) Fluorescence labeling treatment Specimen slides subjected to secondary antibody treatment were subjected to fluorescence labeling treatment by immunostaining in the following procedures (i) to (ii).
(I) The pathological staining particles prepared in Example 4 diluted to 0.02 nM with PBS containing 1% BSA on the specimen slide were placed in a neutral pH environment (pH 6.9 to 7.4) at room temperature. The reaction was carried out for 3 hours under the conditions.
(Ii) The specimen slide after the reaction was washed with PBS.

(2-4) Staining Process for Morphological Observation The specimen slide subjected to the fluorescent labeling process was subjected to the morphological observation staining process (hematoxylin-eosin staining) according to the following procedures (i) to (ii). (I) The slide that was fluorescently labeled with the antibody was stained with Mayer's hematoxylin solution for 5 minutes to perform hematoxylin staining. The slide was then washed with running water at 45 ° C. for 3 minutes.
(Ii) Next, eosin staining was performed by staining with 1% eosin solution for 5 minutes to prepare a stained slide. After the morphological observation staining treatment, 10 staining slides were washed according to the following procedures (i) to (ii).
(I) Ten types of cleaning solutions having pH of 1,2,3,4,5,6,7,8,9 or 10 were prepared. A washing solution having a pH of 1 to 6 was prepared by diluting 1M hydrochloric acid. Pure water was used as the cleaning liquid having a pH of 7. A cleaning solution having a pH of 8 to 10 was prepared by diluting a 28% aqueous ammonia solution.
(Ii) Each of the 10 stained slides subjected to the staining process for morphology observation was immersed in each cleaning solution at room temperature for 10 minutes to perform the cleaning process.

(Dehydration treatment)
Each stained slide subjected to the washing treatment was immersed in dehydrated ethanol for 5 minutes. This operation was repeated 4 times to perform dehydration treatment.

(Solvent replacement process)
Each dyed slide subjected to the dehydration treatment was immersed in xylene at room temperature for 2 to 10 seconds to perform a solvent replacement treatment. This operation was repeated 4 times.

(Encapsulation processing)
The staining slide was encapsulated by the following procedures (i) to (ii).
(I) After the dyeing slide subjected to the solvent replacement treatment was dropped with Enteran New (Merck) at room temperature, it was covered with a cover glass and air-dried at room temperature for 10 minutes to perform an encapsulation treatment.
(Ii) Thereafter, the stained slide subjected to the encapsulation treatment was stored in the dark until the signal was measured.

<Fluorescence image capturing and analysis> was performed in the same manner as described in [0105] above.

* In a table | surface represents the PID score of the dyeing | staining slide produced using the specimen slide of a HER2 positive dyeing | staining control sample. The PID score is an average value of analysis results obtained by counting the number of bright spots of 10 stained slides prepared using specimen slides of HER2-positive stained control specimens derived from the same patient.

Table 3 shows the results of Examples 18 to 27. When an acidic cleaning solution having a pH of 1 to 6 is used, a larger number of pathological staining particles are observed than when a neutral or alkaline cleaning solution having a pH of 7 to 10 is used. It can be seen that the luminance variation (CV) is small.

Claims (19)

1. A method for producing a particle for pathological staining, which comprises producing a particle by condensation polymerization of a compound represented by the following general formula (1).
   

   

   (In Formula (1), R ¹ is each independently a hydrogen atom, CH ₂ OR ² or CH ₂ OH, R ² is an organic group, and at least one of R ¹ is CH ₂ OH. )
2. The method for producing a particle for pathological staining according to claim 1, wherein in the formula (1), at least two of R ¹ are CH ₂ OH.
3. The method for producing particles for pathological staining according to claim 1 or 2, wherein a fluorescent dye is added to and contained in particles obtained by condensation polymerization of the compound represented by the general formula (1).
4. The method for producing particles for pathological staining according to claim 1 or 2, wherein particles containing the fluorescent dye are produced by condensation polymerization of the compound represented by the formula (1) in the presence of the fluorescent dye.
5. The method for producing particles for pathological staining according to claim 3 or 4, wherein the fluorescent dye is rhodamine or an aromatic dye.
6. 6. The particle obtained by condensation polymerization of the compound represented by the formula (1) is reacted with a bifunctional amine to modify the surface of the particle with a group containing an amino group. The manufacturing method of the particle | grains for pathological dyeing of description.
7. The method for producing a particle for pathological staining according to claim 6, wherein the bifunctional amine is methanediamine or polyethylene glycol diamine.
8. The method for producing particles for pathological staining according to any one of claims 3 to 7, wherein the particles for pathological staining are phosphor-integrated nanoparticles, and the average particle diameter thereof is 10 nm to 500 nm.
9. A particle for pathological staining having particles containing a condensation polymer of a compound represented by the following general formula (1).
   

   

   (In Formula (1), R ¹ is each independently a hydrogen atom, CH ₂ OR ² or CH ₂ OH, R ² is an organic group, and at least one of R ¹ is CH ₂ OH. )
10. The pathological staining particle according to claim 9, wherein in the formula (1), at least two of R ¹ are CH ₂ OH.
11. The particle for pathological staining according to claim 9 or 10, wherein the particle for pathological staining is a particle in which a fluorescent dye is contained in a particle containing a polycondensate of the compound represented by the general formula (1).
12. The particle for pathological staining according to claim 11, wherein the fluorescent dye is rhodamine or an aromatic dye.
13. The pathological staining particle according to any one of claims 9 to 12, wherein a group containing an amino group is present on the surface of the particle containing the condensation polymer of the compound represented by the formula (1).
14. The particle for pathological staining according to claim 13, wherein the group containing an amino group present on the surface of the particle has a structure derived from methanediamine or polyethylene glycol diamine.
15. The particle for pathological staining according to any one of claims 9 to 14, wherein the particle for pathological staining is a phosphor-integrated nanoparticle, and an average particle diameter thereof is 10 nm to 500 nm.
16. A process of fluorescently labeling a target biological substance contained in a tissue section on a specimen slide with the pathological staining particles according to any one of claims 9 to 15 by an immunostaining method (fluorescence labeling process),
    A method for washing a stained slide, comprising performing a process of staining the fluorescently labeled tissue section with a staining solution for morphology observation (staining process), and a process of cleaning the stained tissue section with an acidic aqueous solution (cleaning process).
17. The method for washing a stained slide according to claim 16, wherein the pH of the acidic aqueous solution is 2 or more and less than 7.
18. The method for washing a stained slide according to claim 16 or 17, wherein the acidic aqueous solution has a pH of 3 or more and 6.5 or less.
19. The method for washing a staining slide according to any one of claims 16 to 18, wherein the staining solution is selected from hematoxylin and / or eosin.

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