JP6406253B2 - Pathological stain - Google Patents

Description

  The present invention relates to a pathological stain.

  Immunohistochemistry (IHC) is widely known as a histochemical method that uses antibodies to detect substances that can be antigens such as proteins, polysaccharides, or mRNAs present in biological tissues such as pathological tissues. This immunohistochemistry is called “immunostaining” because it performs a color development operation in order to visualize an antigen-antibody reaction that is originally invisible. Due to the feature of visualizing the location of antigen-antibody reaction, immunohistochemistry is widely used in the fields of medicine and biochemistry for the purpose of detecting the location of biopolymers in living tissue.

  In immunostaining, staining methods using enzymes typified by the DAB (diaminobenzidine) method and the like are well known, but this method is greatly influenced by environmental conditions such as temperature or time, It is difficult to accurately measure the target antigen molecule. Therefore, fluorescent dyes with high quantitative performance are used for immunostaining as an alternative labeling reagent, and in recent years, development of fluorescent labels with high brightness has been actively promoted.

  The present inventor has developed a method for performing fluorescent immunostaining on a biological tissue using a fluorescent label composed of fluorescent nanoparticles to which a plurality of fluorescent substances are bonded. Developed a method for quantifying the bright spots observed by irradiating antigen-antibody conjugates with excitation light by adding a pathological staining solution containing fluorescent nanoparticles to which antibodies are bound. (For example, Patent Document 1 and Non-Patent Document 1).

  A pathological staining solution used for fluorescent immunostaining is usually stored at a high concentration, and is diluted when used for fluorescent immunostaining. However, since this pathological stain is a dispersion in which a fluorescent label is dispersed, the particles of the fluorescent label gradually aggregate or settle during storage at rest. For this reason, when fluorescent immunostaining is performed, it is necessary to apply ultrasonic waves to the pathological staining liquid to disperse the aggregated or precipitated particles again, and then dilute the dispersed pathological staining liquid.

  Therefore, it is desired that the pathological staining solution used for fluorescent immunostaining can maintain stable dispersibility. As a countermeasure, it is conceivable to increase the viscosity of the pathological staining solution. However, there is a problem that excessive force is required for the discharge, and that the liquid runs out during discharge.

  Therefore, it was necessary to improve the relationship between the viscosity and dispersibility of the pathological staining liquid dispersion from the viewpoint of solving the above problems and providing a pathological staining liquid that is easy to handle.

JP 2012-194013 A

HER2-Targeted Therapy: Lessons learned and Future Directions, Rita Nahta and Francisco J. Esteva, Clin. Cancer Res., Nov. 2003; 9; 5078-5084

For example, when the pathological staining liquid containing the fluorescent substance-encapsulating silica particles described in Patent Document 1 is stored for about one month, the fluorescent label particles dispersed in the pathological staining liquid aggregate or settle.
An object of the present invention is to provide a pathological staining solution used for fluorescent immunostaining, which comprises a dispersion in which fluorescent nanoparticles do not aggregate or settle even after preparation and after storage for a certain period of time. To do.

  Another object of the present invention is to provide an aqueous dispersion that can be used for the preparation of a pathological staining solution and that does not aggregate or settle the labeled particles even after being stored for a long time.

The present inventor paid attention to the fact that the density of the particles and the dispersion of the fluorescent label and the viscosity of the dispersion affect the aggregation or sedimentation, and as a result of examining these relationships, the formula (1) is satisfied. In some cases, it was found that the particles do not aggregate or settle even after storage for a certain period of time.
Further, the present inventor has paid attention to the fact that the viscosity of the aqueous dispersion affects the aggregation or sedimentation of the labeled particles, and has solved the above problem by imparting thixotropic properties to the aqueous dispersion. The headline and the present invention were completed.

That is, this invention consists of the following matters.
[1] A pathological staining solution comprising a dispersion satisfying the following formula (1), comprising a fluorescent label comprising fluorescent nanoparticles having an average particle size of 50 to 200 nm and a solvent.
(Ρ−ρ _w ) /η≦0.02 (1)
(In the formula (1), ρ represents the solid density (g / cm ³ ) of the fluorescent nanoparticles at 20 ° C., ρ _w represents the density of the dispersion (g / cm ³ ) at 20 ° C., and η is 20 ° C. Represents the viscosity (g / cm · sec) of the dispersion liquid.

  [2] The pathological staining solution according to [1], wherein in the formula (1), the viscosity η (g / cm · sec) of the dispersion is 1 <η <100.

  [3] In addition to the fluorescent label and the solvent, the dispersion further contains a monosaccharide, a disaccharide or an X-ray contrast agent as an additive for adjusting density and / or viscosity, [1] or [2] The pathological staining solution described in 1.

  [4] The pathological staining solution according to [3], wherein the density and / or viscosity adjusting additive is at least one selected from the group consisting of sucrose, glycerol, sorbitol, fructose, and iopamidol.

  [5] An aqueous dispersion containing a particulate marker, a thixotropy-imparting agent, and an aqueous solvent for dispersing or dissolving these two components.

[6] The aqueous dispersion according to [5], wherein the thixotropic agent is a water-soluble polymer.
[7] The aqueous dispersion according to [6], wherein the water-soluble polymer is a carboxymethyl cellulose salt.

[8] The aqueous dispersion according to any one of [5] to [7], which has an apparent viscosity of 10 to 50 mPa · s measured at 25 ° C. and 60 rpm using a B-type viscometer. .
[9] The aqueous dispersion according to any one of [5] to [8], which has a pH of 6 to 8.

[10] The aqueous dispersion according to any one of [5] to [9], wherein the particulate label is a particulate fluorescent label.
[11] The aqueous dispersion according to [10], wherein the particulate fluorescent label is obtained by labeling fluorescent nanoparticles.

[12] The aqueous dispersion according to any one of [5] to [11], wherein the particulate marker is a complex of a biological substance for pathological staining.
[13] A pathological staining solution containing the aqueous dispersion according to [12].
[14] A reagent bottle for an automatic staining apparatus, which is filled with the pathological staining solution according to [12].
[15] The pathological staining solution according to [1], wherein the dispersion is an aqueous dispersion containing a fluorescent label, a thixotropy-imparting agent, and an aqueous solvent for dispersing or dissolving these two components.
[16] The pathological staining solution according to [15], wherein the thixotropic agent is a water-soluble polymer.

The pathological staining solution of the present invention does not aggregate or settle particles such as fluorescent labels in the dispersion even after being stored for a certain period (for example, one month) after preparation. It can be used for fluorescent immunostaining without performing an operation of dispersing the particles.
The pathological staining liquid of the present invention can be easily sucked up with a pipette, and is easy to handle because the pathological staining liquid does not remain in the pipette when discharged.

  In addition, according to the present invention, by adding a thixotropy-imparting agent to an aqueous dispersion to increase the viscosity during storage, a pathological staining solution is prepared in which sedimentation or aggregation of a label such as fluorescent nanoparticles is suppressed. can do. By stably dispersing the label in the pathological staining solution without sedimentation or aggregation, individual labels can be bound to the target molecule on the living tissue without staining, and the number of bright spots can be measured. In addition, the number of molecules to be stained can be accurately evaluated. Further, such a pathological staining solution is applied with a shearing force due to ejection during use, so that its viscosity is lowered and can be placed evenly on a living tissue.

FIG. 1 is a schematic view showing a state in which a pathological staining solution is discharged onto a living tissue from a reagent bottle of an automatic staining apparatus. In the prior art, since the fluorescently labeled particles aggregate or settle in the pathological staining solution, the discharge amount and concentration become unstable, and when the particles aggregate or settle, the distribution of the particles on the living tissue becomes non-uniform. In contrast, the number of bright spots cannot be measured accurately, but in the present invention, the dispersibility of the pathological staining solution is maintained, so that sedimentation and aggregation are prevented, and the fluorescent label can be uniformly placed on the living tissue. FIG. 2 shows a case where the aqueous dispersion (pathological staining liquid) measured in Comparative Example 5-1 and Example 5-1 is discharged five times from the reagent bottle of the automatic staining apparatus while setting the discharge amount to 150 μL. It is a graph which shows the fluctuation | variation of discharge amount (change rate [%] of the discharge amount of each time when the discharge amount of the 1st time is used as a reference). In Comparative Example 5-1, in which no thixotropy-imparting agent was added, the discharge amount was not constant, whereas in Example 5-1, where agar, a thixotropy-imparting agent was added, the aqueous dispersion was stably ejected. I understand. FIG. 3 shows a luminance histogram (indicated by the number of bright spots on the vertical axis and the horizontal axis on the horizontal axis) when an aqueous dispersion (pathological staining liquid) was discharged onto an APS-coated slide glass, as measured in Comparative Example 6-1 and Example 6-1. Brightness). In Comparative Example 6-1 in which no thixotropy-imparting agent was added, the luminance histogram showed a broad distribution due to aggregation of fluorescent labeling particles, whereas in Example 7-1 in which the thixotropy-imparting agent xanthan gum was added, It can be seen that the aggregation of the fluorescent label particles is suppressed in the dispersion, and the luminance histogram distribution is narrow. FIG. 4 is a photographed image with a fluorescence microscope showing the state of the bright spot of the fluorescent label bonded to the antigen on the living tissue.

The pathological staining solution of the present invention will be described in detail below.
[Fluorescent nanoparticles]
The pathological staining solution of the present invention comprises a dispersion containing a fluorescent label composed of fluorescent nanoparticles having an average particle size of 50 to 200 nm and a solvent.

  The fluorescent nanoparticles used in the present invention may be any existing one as long as it is a label for immunostaining and can be used for immunostaining. For example, the fluorescent nanoparticles used in the present invention may be any of (A) inorganic fluorescent nanoparticles, (B) fluorescent dye-containing nanoparticles, and (C) fluorescent nanoparticle-containing particles described below.

  The fluorescent nanoparticles are particles having an average particle size of 50 to 200 nm, preferably 80 to 150 nm. When the average particle diameter of the fluorescent nanoparticles exceeds 200 nm, the number of bright spots may decrease due to a decrease in reaction efficiency. When the average particle diameter of the fluorescent nanoparticles is less than 50 nm, the brightness decreases due to a decrease in luminance. The score may decrease.

The coefficient of variation indicating the variation in the average particle diameter of the fluorescent nanoparticles is not particularly limited, but is usually 15% or less, preferably 10% or less.
The average particle diameter of the fluorescent nanoparticles is obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of the fluorescent nanoparticles, and taking the measured value as the area of the corresponding circle. It can be measured as a diameter (area circle equivalent diameter). The average (particle size) and coefficient of variation of the particle size of the population of fluorescent nanoparticles are determined after measuring the particle size (particle size) as described above for a sufficient number (for example, 1000) of fluorescent dye-containing nanoparticles. The average particle diameter is calculated as the arithmetic average thereof, and the coefficient of variation is calculated by the formula: 100 × standard deviation of particle diameter / average particle diameter.

(A) Inorganic fluorescent nanoparticles The inorganic fluorescent nanoparticles include semiconductor nanoparticles and nanoparticles composed of other inorganic phosphors.

Examples of the semiconductor include II-VI group semiconductors such as ZnSe, ZnTe, CdSe, CdTe, PbS, PbSe, and PbTe, and II-VI group semiconductors such as AlAs, AlSb, GaP, GaAs, GaSb, InP, InAs, and InSb. From the viewpoint of toxicity, GaP and InP can be preferably used. These can be used alone or in combination.
As other inorganic phosphors, for example, Y ₂ O ₃ , YVO ₄ , ZnO, ZnS or the like can be used as a base material, and Eu or Nd can be used alone or in combination at the emission center.

(B) Fluorescent dye-encapsulated nanoparticles Fluorescent dye-encapsulated nanoparticles are nano-sized particles having a structure in which a plurality of fluorescent dyes are encapsulated in particles (matrix) made of an organic substance or an inorganic substance. The fluorescent dye-containing nanoparticles used in the present invention can be prepared by a known method after selecting an appropriate fluorescent dye and an organic or inorganic material that forms the particles.

  Examples of organic or inorganic substances that form particles include polystyrene, polyamide, polylactic acid, polyacrylonitrile, polyglycidyl methacrylate, melamine resin, polyurea, polybenzoguanamine, polyfuran, polyxylene, phenol resin, polysaccharide, silica, and the like. The thing which can include fluorescent dye is mentioned. Examples of the melamine resin include a water-soluble melamine resin “Nicalac MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.).

  The encapsulated fluorescent dye is selected from, for example, rhodamine dye molecules, squarylium dye molecules, cyanine dye molecules, aromatic ring dye molecules, oxazine dye molecules, carbopyronine dye molecules, and pyromesene dye molecules. can do. Alternatively, Alexa Fluor (registered trademark, manufactured by Invitrogen) dye molecule, BODIPY (registered trademark, manufactured by Invitrogen) dye molecule, Cy (registered trademark, manufactured by GE Healthcare) dye molecule, DY (registered trademark, DYOMICICS) Manufactured) dye molecule, HiLyte (registered trademark, manufactured by Anaspec) dye molecule, DyLight (registered trademark, manufactured by Thermo Scientific) dye molecule, ATTO (registered trademark, manufactured by ATTO-TEC) dye molecule MFP (registered trademark, manufactured by Mobitec) type dye molecule, and the like. The generic names of these dye molecules are named based on the main structures (skeletons) in the compound or registered trademarks, and the range of fluorescent dyes belonging to each is appropriate for those skilled in the art without undue trial and error. Can be grasped.

  Specific examples of rhodamine dye molecules include 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, Texas Red, Spectrum Red (Spectrum Red). ), LD700 PERCHLORATE, and the like.

  Specific examples of the squarylium dye molecule include SRfluor, 680-carboxylate, 1,3-bis [4- (dimethylamino) -2-hydroxyphenyl] -2,4-dihydroxycyclobutenediylium dihydroxide (1,3 -bis [4- (dimethylamino) -2-hydroxyphenyl] -2,4-dihydroxycyclobutenediylium dihydroxide), bis, 1,3-bis [4- (dimethylamino) phenyl] -2,4-dihydroxycyclobutenediylium dihydroxide (Bis, 1,3-bis [4- (dimethylamino) phenyl] -2,4-dihydroxycyclobutenediylium dihydroxide) bis (2- (4- (diethylamino) -2-hydroxyphenyl) -4- (4- (diethyliminio) ) -2-Hydroxycyclohexa-2,5-dienylidene) -3-oxocyclobute-1-enolate (bis, 2- (4- (diethylamino) -2-hydroxyphenyl) -4- (4- (diethyliminio) -2- hydroxycyclohexa-2,5-dienylidene)- 3-oxocyclobut-1-enolate), 2- (4- (dibutylamino) -2-hydroxyphenyl) -4- (4- (dibutyliminio) -2-hydroxycyclohexa-2,5-dienylidene) -3-oxocyclobute -1-enolate (2- (4- (dibutylamino) -2-hydroxyphenyl) -4- (4- (dibutyliminio) -2-hydroxycyclohexa-2,5-dienylidene) -3-oxocyclobut-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- Oxocyclobute-1-enolate (2- (8-Hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido [3,2,1-ij] quinolin-9-yl )-Four- (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).

  Specific examples of cyanine dye molecules include 1-butyl-2- [5- (1-butyl-1,3-dihydro-3,3-dimethyl-2H-indole-2-ylidene) -penta-1,3. -Dienyl] -3,3-dimethyl-3H-indolium hexafluorophosphate (1-butyl-2- [5- (1-butyl-1,3-dihydro-3,3-dimethyl-2H-indol-2- ylidene) -penta-1,3-dienyl] -3,3-dimethyl-3H-indolium hexafluorophosphate), 1-butyl-2- [5- (1-butyl-3,3-dimethyl-1,3-dihydro-) Indole-2-ylidene) -3-chloropenta-1,3-dienyl] -3,3-dimethyl-3H-indolium hexafluorophosphate (1-butyl-2- [5- (1-butyl-3,3- dimethyl-1,3-dihydro-indol-2-ylidene) -3-chloropenta-1,3-dienyl] -3,3-dimethyl-3H-indolium hexafluorophosphate), 3-ethyl-2- [5- (3- Ethyl H-benzothioazole-2-ylidene) -penta-1,3-dienyl] -benzothiazole-3-ium-iodide (3-ethyl-2- [5- (3-ethyl-3H-benzothiazol-2-ylidene) ) -penta-1,3-dienyl] -benzothiazol-3-ium iodide).

Specific examples of the aromatic ring dye molecule include N, N-bis- (2,6-diisopropylphenyl) -1,6,7,12- (4-tert-butylphenoxy) -perylene-3,4,9. , 10-Tetracarboxylic acid diimide (N, N-bis- (2,6-diisopropylphenyl) -1,6,7,12- (4-tert-butylphenoxy) -perylen-3,4,9,10-tetracarboxylic acid diimide), N, N′-bis (2,6-diisopropylphenyl) -1,6,7,12-tetraphenoxyperylene-3,4: 9,10-tetracarboxydiimide (N, N′-bis (2 , 6-diisopropylphenyl) -1,6,7,12-tetraphenoxyperylene-3,4: 9,10-tetracarboxdiimide), N, N′-bis (2,6-diisopropylphenyl) perylene-3,4,9,10 -Bis (dicarboimide) (N, N'-bis (2,6-diisopropylphenyl) perylene-3,4,9,10-bis (dicarbimide)), 16N, N'-bis (2,6-dimethyl) Phenyl) perylene-3,4,9,10-tetracarboxylic diimide (16N, N'-bis (2,6-dimethylphenyl) perylene-3,4,9,10-tetracarboxylic diimide), 4,4 '-[ (8,16-dihydro-8,16-dioxodibenzo [a, j] perylene-2,10-diyl) dioxy] dibutyric acid (4,4 '-[(8,16-dihydro-8,16-dioxodibenzo [a, j] perylene-2,10-diyl) dioxy] dibutyric acid), 2,10-dihydroxy-dibenzo [a, j] perylene-8,16-dione (2,10-dihydroxy-dibenzo [a, j ] perylene-8,16-dione), 2,10-bis (3-aminopropoxy) 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 (3 , 3 '-[(8,16-dihydro-8,16-dioxodibenzo [a, j] perylen-2,10-diyl) dioxy] dipropyl amine]), 17-bis (octyloxy) anthra [9,1,2-cde-] benzo [rst] pentaphen-5-10-dione (17-bis (octyloxy) anthra [9,1,2-cde- ] benzo [rst] pentaphene-5-10-dione), octadecanoic acid, 5,10-dihydro-5,10-dioxoanthra [9,1,2-cde] benzo [rst] pentaphen-16,17-diyl Esters (octadecanoic acid, 5,10-dihydro-5,10-dioxoanthra [9,1,2-cde] benzo [rst] pentaphene-16,17-diyl ester), dihydroxydibenzanthrone, benzenesulfonic acid , 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-tetra Ru] tetrakis (oxy)] tetrakis-, benzeneethaneaminium (benzenesulfonic acid, 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-, benzeneethanaminium), 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-] (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 the like.

Specific examples of the oxazine dye molecule include Cresyl violet, Oxazine 170, EVOblue30, and Nile Blue.
Specific examples of carbopyronine dye molecules include CARBOPYRONIN 149.

Specific examples of the pyromesenic dye molecule include PYRROMETHENE 650.
Specific examples of Alexa Fluor dye molecules include Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 Alexa Fluor 750 and the like (manufactured by Invitrogen).

  Specific examples of BODIPY dye molecules include BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650 And BODIPY 650/665 (manufactured by Invitrogen).

Specific examples of the Cy dye molecule include Cy3.5, Cy5, Cy5.5 (above, manufactured by GE Healthcare) and the like.
Specific examples of DY dye molecules include DY-590, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633, DY-634 (above, manufactured by DYOMICICS), etc. Is mentioned.

Specific examples of the HiLyte dye molecule include HiLyte Fluor 594, HiLyte Fluor TR (manufactured by Anaspec) and the like.
Specific examples of the DyLight-based dye molecule include DyLight 594, DyLight 633 (manufactured by Thermo Scientific).

Specific examples of ATTO dye molecules include ATTO590, ATTO610, ATTO620, ATTO633, ATTO655, etc. (above, manufactured by ATTO-TEC).
Specific examples of MFP dye molecules include MFP590, MFP631 (manufactured by Mobitec) and the like.

  Examples of other dyes include C-phycocyanin, phycocyanin, APC (allophycocyanin), APC-XL, Northern Lights 637 (manufactured by R & D Systems), and the like.

Moreover, these derivatives (what can function as a fluorescent dye, for example, a well-known derivative) can be mentioned.
The fluorescent dyes as described above may be included in the fluorescent dye-containing nanoparticles in a single kind or in a mixture of a plurality of kinds.

  For example, fluorescent dyes such as rhodamine dye molecules and aromatic ring dye molecules are preferred because of their relatively high light resistance, and among them, perylene belonging to aromatic ring dye molecules, particularly perylene diimide, is preferred. On the other hand, even if the fluorescent dye has relatively low light resistance, there is a possibility that fluorescent dye-containing nanoparticles satisfying the predetermined luminance retention rate according to the present invention can be produced by selecting an appropriate matrix.

  The method for producing the fluorescent dye-containing nanoparticles is not particularly limited. Any method may be used for introducing the dye into the particle, such as a method of synthesizing the particle by bonding a dye molecule to the monomer as the particle raw material, or a method of introducing the dye by adsorbing the dye to the particle.

  Here, as the monomer, a (co) monomer having a predetermined functional group in the side chain in advance is (co) polymerized, or after the preparation of the organic or inorganic particles (matrix), There is a method in which a bound functional group is treated with a reagent and converted to the predetermined functional group.

  Specifically, an embodiment in which polystyrene resin particles having an epoxy group on the surface are produced by copolymerization using glycidyl methacrylate as a monomer together with styrene, or copolymerization using melamine and formaldehyde as monomers. The embodiment etc. which manufacture melamine system resin by are mentioned. In addition, the epoxy group which glycidyl methacrylate has can also be converted into an amino group by a predetermined reagent treatment.

(C) Inorganic fluorescent nanoparticle-encapsulated particles The inorganic fluorescent nanoparticle-encapsulated particles used in the present invention include the inorganic fluorescent nanoparticles described in (A) above for the organic or inorganic particles (matrix). It will be. The inorganic fluorescent nanoparticles may be included in the fluorescent dye-encapsulated nanoparticles alone or in a mixture of a plurality of types.

The method for producing the fluorescent dye-containing nanoparticles is not particularly limited. Use any method to introduce inorganic fluorescent nanoparticles into particles, such as the method of synthesizing particles by bonding inorganic fluorescent nanoparticles to the above monomer, which is the raw material of the particles, or the method of adsorbing and introducing inorganic fluorescent nanoparticles to the particles. It doesn't matter.
Of the (A) inorganic fluorescent nanoparticles, (B) fluorescent dye-containing nanoparticles, and (C) inorganic fluorescent nanoparticle-containing particles, (B) fluorescent dye-containing nanoparticles are preferable.

When immunostaining a fluorescent-labeled specific antigen, a label (conjugate) in which fluorescent nanoparticles and a primary antibody are linked is prepared and directly bound to the antigen (primary antibody method); A method in which a labeled body in which fluorescent nanoparticles and a secondary antibody are linked is prepared and bound to a primary antibody bound to an antigen (secondary antibody method); a labeled body in which fluorescent nanoparticles and avidin or streptavidin are linked The biotin of the biotin-modified primary antibody bound to the antigen or the biotin of the biotin-modified secondary antibody bound to the primary antibody or the biotin and avidin or streptavidin in these binding modes were replaced. Method (biotin-avidin method).

  The fluorescent nanoparticles of the present invention are used in the form of a label as described above based on these methods. Here, the fluorescent nanoparticles are stored as a dispersion in a non-labeled state, and immediately before immunostaining, the fluorescent nanoparticles and a reagent for labeling are reacted to form a label. Alternatively, a pathological staining solution may be prepared, but it is preferable to store it as a dispersion in a labeled state so that it can be used immediately when immunostaining is performed. That is, the fluorescent nanoparticles used in the present invention are preferably labeled in advance, and the pathological staining solution according to the present invention is composed of a dispersion of such labeled fluorescent nanoparticles, or the dispersion It is defined as containing the liquid and further ingredients added as needed. In this case, the fluorescent nanoparticles are preferably combined with avidin or streptavidin so that the use of the pathological staining solution (antigen to be immunostained) is not limited. .

  When fluorescent nanoparticles are complexed, the density and viscosity of the dispersion are measured in a state containing such complexed fluorescent nanoparticles, while the solid density of the fluorescent nanoparticles is measured. Let the solid density of the uncomplexed fluorescent nanoparticles be taken as the solid density of the composited fluorescent nanoparticles (the difference between these solid densities is very small).

  Any primary antibody may be used for immunostaining, and it varies depending on the subject to be immunostained. For example, when performing immunostaining using Ki67 as an antigen, an anti-Ki67 antibody is used. Any secondary antibody may be used, and varies depending on the primary antibody. For example, anti-mouse, rabbit, cow, goat, sheep, dog, chicken antibody can be mentioned.

  Any existing method may be used for binding the fluorescent nanoparticles to the antibody or biotin. For example, amidation by reaction of amine and carboxylic acid, sulfidation by reaction of maleimide and thiol, imination by reaction of aldehyde and amine, amination by reaction of epoxy and amine can be used.

  Antigens that can be the target of immunostaining include HER-2, HER-3, HER-4 (Human Epidermal Growth Factor Receptor), EGFR (Epidermal Growth Factor Receptor) in addition to Ki67 described above. (Epidermal growth factor receptor)), PDGFR (Platelet-Derived Growth Factor (platelet-derived growth factor receptor)), VEGRR (Vascular Endothelial Growth Factor Receptor (vascular endothelial growth factor receptor)), NGFR (Nerve Growth Factor Receptor) (Nerve growth factor receptor)), FGFR (Fibroblast Growth Factor Receptor (fibroblast growth factor receptor)), IR (Insulin Receptor (insulin receptor)), ER (Estrogen Receptor (estrogen receptor)), PgR ( Progesterone Receptor (progesterone receptor)), c-Met (hepatocyte growth factor receptor), TNF-α (Tumor Necrosis Factor (tumor) Necrosis factor)) receptor, IL-6 (Interleukin) receptor, c-Kit (stem cell factor receptor), ALK (Anaplastic lymphoma kinase), etc. Receptors expressed on the cell surface for biological substances: CD31 (PECAM-1) (Platelet Endothelial Cell Adhesion Molecule-1), CD34 (Endothelial cell marker (endothelial cell marker)), GPC3 (Glypican (Glypican) 3) and other marker molecules expressed on the cell surface (glycoprotein etc.); CK7 (Cytokeratin (cytokeratin)), Actin (actin), p53 and other molecules expressed in cells; RSVF protein, B Hepatitis B surface antigen, hepatitis B virus core antigen, hepatitis C virus core antigen, NS3 (Non-s Examples include molecules expressed in viruses such as tructural protein (nonstructural protein) 3). Since these antigens have a high expression level in specific viruses or cells (for example, cancer cells, leukocytes), they can be used as targets for detecting them.

  The particulate label may be contained in the aqueous dispersion at a concentration usually used according to the purpose of immunostaining, and is usually 0.01 to 2.00 mmol, preferably 0.04 to 1.00 mmol with respect to 1 mL of the aqueous dispersion. Add at a rate of When the amount of the particulate label in the aqueous dispersion is within the above range, even if the aqueous dispersion is placed in a reagent bottle in the automatic staining apparatus and stored for a long time, the dispersibility of the particles is maintained well. .

[Thixotropic agent]
As the thixotropy imparting agent in the present invention, any existing thixotropy may be used as long as it can impart thixotropy to the aqueous dispersion. In general, substances known as thickening polysaccharides, thickeners, emulsion stabilizers and the like can be used as the thixotropic agent in the present invention.

Here, the thixotropic property means a property that the viscosity is high at a low shear rate and the viscosity is lowered at a high shear rate.
Examples of the thixotropic agent include water-soluble polymers such as xanthan gum, welan gum, succinoglycan, guar gum, locust bean gum, tamarind gum, pectin and derivatives thereof, carboxymethylcellulose (CMC) salts, hydroxyethylcellulose, alginates, Glycomannan, agar, carrageenan, etc., thickening polysaccharides having gelling ability; polymers having a molecular weight of 100,000 to 150,000 mainly composed of alkyl methacrylate and synthetic resins such as crosslinkable acrylic acid polymers, PEG Nonionic thickeners (surfactants) of HLB8-12.

  Among these, water-soluble polymers such as xanthan gum, guar gum, carboxymethyl cellulose (CMC) salt, and PEG-based nonionic thickener are more preferable from the viewpoint of dispersion stability. When a carboxylate or a sulfonate is formed like a carboxymethyl cellulose (CMC) salt, a monovalent salt such as a sodium salt, a potassium salt, a lithium salt, or an ammonium salt is more preferable.

  The thixotropy-imparting agent is usually added in an amount of 1 to 800 mg, preferably 1 to 100 mg, in 1 mL of an aqueous dispersion. When the amount of the thixotropy-imparting agent in the aqueous dispersion is within the above range, even if the aqueous dispersion is placed in a reagent bottle in the automatic staining apparatus and stored for a long time, a label for pathological staining (for example, a fluorescent dye) The dispersibility of the encapsulated fluorescent nanoparticles) is maintained well.

[solvent]
The solvent used in the present invention may be any existing solvent as long as it can be used for immunostaining, and preferably can be adjusted to a predetermined viscosity by dissolving a viscosity adjusting additive. In general, a buffer solution such as water (pure water) or PBS (phosphate buffer physiological saline) is used.

[Aqueous solvent]
The aqueous dispersion of the present invention contains an aqueous solvent for dispersing or dissolving these two components in addition to the particulate marker and the thixotropic agent. As the aqueous solvent, any existing solvent may be used as long as it can be used for immunostaining, specifically, a thixotropy-imparting agent can be dissolved and adjusted to a predetermined viscosity. In general, a buffer solution such as water (pure water) or PBS (phosphate buffer physiological saline) is used. In consideration of biocompatibility and transparency required for pathological diagnosis, the solvent blended in the aqueous dispersion of the present invention consists essentially of an aqueous solvent and does not contain an organic solvent (aqueous dispersion) It is preferable to mix organic solvents that are difficult to completely eliminate, such as those used at the time of preparation, attached to various components to be blended in, but intentionally do not add organic solvents) .

[Water dispersion]
The aqueous dispersion of the present invention contains the particulate labeling body, the thixotropic agent, and the aqueous solvent as described above. Such an aqueous dispersion is suitable mainly for use as a pathological staining solution as described later, or for the preparation of a pathological staining solution, but takes advantage of the property that the particulate labeling substance is difficult to precipitate and aggregate. It can also be used in other applications.

The apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion of the present invention at 25 ° C. is preferably 10 to 200 mPa · s, more preferably 10 to 50 mPa · s.
Here, in the present invention, the apparent viscosity refers to the flow characteristics of the aqueous dispersion, using a B-type viscometer, measuring the torque when the rotor is rotated at a predetermined rotational speed, and measuring the shear rate and shear. The relationship of stress (shear stress / shear rate) is obtained.

A method for measuring the apparent viscosity of a 1% xanthan gum aqueous solution using a B-type viscometer will be described with a specific example.
About 2.2 g of xanthan gum is precisely weighed into a 300 mL Erlenmeyer flask with a stopper, and dissolved water is added according to the following formula.
Dissolved water (g) = xanthan gum (g) × (99-water content (%))

The resulting aqueous solution is left overnight and stirred with a magnetic stirrer for about 5 minutes to form a complete solution, which is then transferred to a container with a lid having a diameter of about 45 mm and a height of about 145 mm, and a constant temperature bath at 25 ± 0.2 ° C. After 30 minutes, gently stir the solution with a glass rod, attach the rotor and guard to the B type viscometer (BII type viscometer), rotate the rotor, and after 3 minutes, mark the B type viscometer. read. According to the type of rotor (No. 1 to 4) and the rotational speed, the coefficient in Table 1 is multiplied to obtain the viscosity. In addition, the kind and rotation speed of a rotor are selected according to the level of the viscosity of a xanthan gum aqueous solution.
Viscosity (mPa · s) = Reading scale × Coefficient

  If the apparent viscosity of the aqueous dispersion exceeds the above range, when adding the aqueous dispersion onto the living tissue using an automatic staining device, the aqueous dispersion may have a high viscosity and may not be discharged in an accurate amount. Below the above range, the particles may aggregate or settle in the aqueous dispersion, and it may not be possible to uniformly place the label on the living tissue.

  The pH of the aqueous dispersion of the present invention is preferably 6-8, more preferably around 7. When the pH deviates from neutrality, that is, when the pH is inclined toward the acidic side or the basic side, the number of bright spots on the living tissue after discharging the aqueous dispersion onto the living tissue is reduced as compared with the neutral side. This is because when the pH is acidic or basic, the antigen-antibody reaction is less likely to proceed, for example, the reaction between the avidin complex and the biotin-labeled protein is less likely to occur than in the neutral case. This is because the dyeability is lowered.

The density of the aqueous dispersion of the present invention is usually 1.00 to 3.00 g / cm ³ , preferably 1.00 to 2.00 g / cm ³ . When the density of the aqueous dispersion exceeds the above range, the movement of particles in the aqueous dispersion may be suppressed, and the staining property of immunostaining may be reduced.

  The aqueous dispersion of the present invention is preferably transparent. If the aqueous dispersion contains solid components such as impurities, the number of bright spots on the biological tissue is reflected when the excitation light is irradiated after the aqueous dispersion is discharged onto the biological tissue. May not be measured accurately.

[Dispersion]
The dispersion used in the present invention is obtained by dispersing a fluorescent label comprising the fluorescent nanoparticles in the solvent.

  The dispersion liquid only needs to be dispersed at a concentration that is usually used according to the purpose of immunostaining. For example, the label formed of the fluorescent nanoparticles is usually 0.02-1 to 1 mL with respect to 1 mL of the solvent. Disperse at a rate of 2 nM, preferably 0.3 nM.

The dispersion satisfies the following formula (1).
(Ρ−ρ _w ) /η≦0.02 (1)
In the above formula (1), ρ represents the solid density (g / cm ³ ) of the fluorescent nanoparticles at 20 ° C., ρ _w represents the density of the dispersion at 20 ° C. (g / cm ³ ), and η is 20 ° C. Represents the viscosity (g / cm · sec) of the dispersion liquid.

Here, the solid density ρ (g / cm ³ ) of the fluorescent nanoparticles at 20 ° C. is measured using a density gradient centrifugation method.
The density ρ _w (g / cm ³ ) of the dispersion at 20 ° C. is measured by taking a predetermined volume of the dispersion in a volumetric flask and weighing the weight.

The viscosity η (g / cm · sec) of the dispersion at 20 ° C. is measured using a tuning fork type vibration viscometer.
The dispersion satisfying such conditions is preferably the same as that of the fluorescent labeling particles and solvent to be used, and the type (property) and amount of the later-described density and / or viscosity adjusting additive used as necessary. It can be prepared by adjusting within the range as described in. For example, when using a label composed of fluorescent nanoparticles with a relatively low solid density ρ (close to the specific gravity of water), the difference from the density ρ _{w of the} dispersion (that is, the molecule on the left side of equation (1)) is compared. Therefore, it is easy to prepare a dispersion satisfying the formula (1) without increasing the viscosity η of the dispersion too much. Conversely, when using fluorescent nanoparticles with a relatively large solid density ρ, the difference from the density ρ _{w of the} dispersion becomes relatively large. For example, using a highly thickening additive, the viscosity of the dispersion When η is increased, a dispersion satisfying the formula (1) can be easily prepared.

In the above formula (1), the viscosity η (g / cm · sec) of the dispersion is preferably 1 <η <100, and more preferably 1 <η <30.
When the dispersion satisfies the relationship of the above formula (1), after preparing a pathological staining solution composed of the dispersion, the fluorescent nanoparticles are stored in the dispersion after being stored at 4 ° C. for a certain period (for example, about one month). Since particles such as these do not aggregate or settle, the pathological staining solution can be immediately diluted and applied to the living tissue without the operation of dispersing the particles by applying ultrasonic waves before dilution. Further, when applied to a pathological tissue, the pathological stain is sucked up and discharged by a pipette. At this time, since the pathological stain does not remain in the pipette, the handling is easy.

  The pathological staining solution of the present invention is prepared by dispersing a label comprising the fluorescent nanoparticles in the solvent. The preparation method is not particularly limited, and after adding fluorescent nanoparticles in a solvent at room temperature, the mixture is stirred by pipetting using a pipetman or the like.

The pathological staining solution of the present invention may further contain a density and / or viscosity adjusting additive (hereinafter also simply referred to as “additive”) in addition to the fluorescent nanoparticles and the solvent in the dispersion.
The additive may be any one that can be used for immunostaining and can adjust density and / or viscosity. For example, sucrose, glycerol, sorbitol, fructose, X-ray contrast agents such as mannitol, galactose, mannose, ribose, deoxyribose, galactose, trehalose, etc., or monosaccharides or disaccharides (sugar alcohols) or iopamidol, iodixanol, iohexol, iotrolane, iomeprol, ioversol, iopromide, etc. It is done. Of these, sucrose, glycerol, sorbitol, fructose, iopamidol and the like are preferable. Any one of these additives may be used alone, or a plurality of these additives may be used in combination as necessary.

The additive can be added so that the relationship between the viscosity and dispersibility of the dispersion of the pathological staining solution satisfies the relationship of the above formula (1), for example, generally 0.6 to 0 with respect to 1 mL of the solvent. Add at a rate of 9 g. The amount of the additive added is appropriately adjusted according to the type so that the density of the dispersion is in the predetermined range as described above, and preferably the viscosity of the dispersion is further in the predetermined range as described above. can do.
The method of adding the additive is not particularly limited, and the additive may be added to the dispersion at room temperature and, if necessary, stirred by pipetting using a pipetman or the like.

[Pathological staining solution]
Method for Using Pathological Staining Solution The pathological staining solution of the present invention is used for immunostaining. An example of the method of immunostaining is shown.

  The immunostaining method is the same as a general biological substance detection method. Usually, a pathological tissue is placed on a base glass treated with an amino group-containing silane coupling agent, deparaffinized, and then a blocking agent is added. And the step (ii) of adding the pathological staining solution onto the pathological tissue, reacting the antibody with the antigen in the pathological tissue, and immunostaining.

  The base glass treated with the amino group-containing silane coupling agent used in step (i) is, for example, an adhesive between a pathological tissue section and a glass surface, such as an aminosilane-coated slide glass. A silane-coated product. The base glass treated with the amino group-containing silane coupling agent is hereinafter simply referred to as “aminosilane-coated slide glass”.

  The aminosilane-coated slide glass can be produced, for example, by coating the slide glass with aminopropyltriethoxysilane, aminopropyltrimethoxysilane, aminopropylmethyldimethoxysilane, or the like. Commercially available products such as S08110 (APS (Aminosilane) coated slide glass manufactured by Matsunami Glass Industrial Co., Ltd.) and Silane 1106 (An anti-peeling agent coated slide manufactured by Muto Chemical Co., Ltd.) may be used for the aminosilane coated slide glass.

  Deparaffinization is a process that involves immersing an aminosilane-coated slide glass with a pathological tissue in a sufficient amount of deparaffinizing agent placed in a glass container, etc. It is to be removed from the tissue. The immersion may be repeated a plurality of times by replacing the deparaffinizing agent with a clean one, or changing the deparaffinizing agent for each container. As the deparaffinizing agent, xylene is usually used.

  As the blocking agent, artificial synthetic polymers, animal sera such as normal serum, rabbit serum, goat serum and rat serum, and existing ones such as bovine serum albumin, gelatin and casein are used without particular limitation. Of these, animal serum and bovine serum albumin are preferable, and animal serum is more preferable.

  The blocking agent is added to the pathological tissue fixed on the aminosilane-coated slide glass. The appropriate amount of the blocking agent added to the pathological tissue may be an amount that can cover the pathological tissue, and is usually 30 to 800 μL, preferably 50 to 300 μL.

  For the pathological tissue, for example, a pathological section containing a non-self substance such as cancer is used. Specifically, a tissue obtained by slicing a tissue such as breast cancer into a thickness of about 1 to 20 μm is used. Further, for example, a commercially available product such as a liver cancer tissue slide (T031 manufactured by US Biomax) may be used. This liver cancer tissue slide is generally used as a sample of liver cancer.

  Step (ii) is an immunostaining step. That is, for example, the pathological staining solution of the present invention diluted about 5 times with PBS is added onto a pathological tissue section, the pathological tissue to be detected is stained, and then an encapsulant is added, and then a cover glass To make an evaluation slide.

  The immunostaining described above is not limited to tissue staining, and can also be applied to cell staining. The pathological tissue material to be detected is not particularly limited as long as a substance that specifically binds to the fluorescent label exists. Typically, a combination of an antigen and an antibody is used as described above. For example, a combination of a nucleic acid molecule (oligonucleotide, polynucleotide) and a nucleic acid molecule having a sequence capable of complementary binding thereto can be used. It is.

The tissue section subjected to immunostaining is dehydrated and permeated with an organic solvent, and then encapsulated with an encapsulating agent.
Dehydration and penetration are performed by washing the stained tissue section with an aqueous washing solution such as PBS (phosphate buffered saline), followed by dehydration with ethanol and replacement with xylene. Dehydration with ethanol is achieved by immersing tissue sections in ethanol with a reduced water content such as 50%, 30%, 10%, and 0%, and replacing it with ethanol. Do. By immersing the ethanol-substituted section in xylene, xylene replacement is performed, and the section is penetrated. Encapsulation is performed by placing an encapsulant on the xylene-substituted section and placing a cover glass or the like.
The encapsulating agent is preferably an oil-based encapsulating agent, and examples thereof include commercial products such as Cosmo Bio's Mount Quick and Merck's Enteran New.

[Pathological staining solution]
-Method for Using Pathological Staining Solution The pathological staining solution of the present invention can be prepared as containing an aqueous dispersion as described above. That is, the aqueous dispersion of the present invention as described above may be used as a pathological staining liquid as it is, or a pathological staining liquid is prepared by further adding an additive to the aqueous dispersion according to the use for pathological staining. May be.

The pathological staining solution of the present invention is preferably used for an automatic staining apparatus, that is, for filling a reagent bottle that is set and used in the automatic staining apparatus.
An example of a method of immunostaining using a pathological stain is shown.

Fluorescence observation By irradiating the evaluation slide obtained by the above process with excitation light having a predetermined wavelength (for example, excitation wavelength 575-600 nm, fluorescence wavelength 612-682 nm), the fluorescence emitted by the fluorescent nanoparticles is observed. . Thereby, a predetermined biomolecule existing in the pathological tissue can be detected.

  For irradiation of excitation light, it is only necessary to use the same irradiation means as in general fluorescence observation, for example, from a laser light source provided in a fluorescence microscope, using a filter that selectively transmits a predetermined wavelength as necessary, What is necessary is just to irradiate the dye | stained tissue section with the excitation light of a suitable wavelength and output.

  The observation of fluorescence may be performed from a lens barrel of a fluorescence microscope, or may be performed by separately displaying an image captured by a camera installed on the fluorescence microscope on a display unit such as a monitor. Moreover, you may use the filter which selectively permeate | transmits a predetermined wavelength as needed.

Specifically, the number of bright spots present in the pathological tissue placed on the aminosilane-coated slide glass is measured. The area of the nucleus in the entire observation field and the number of bright spots existing in the nucleus are measured, the number of bright spots per unit area (pieces / μm ² ) is calculated, and the average value in the three fields is obtained to calculate as a signal.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
[Example 1-1]
(1) Preparation of pathological stain
Preparation of antibody-bound fluorescent melamine resin particles (average particle size 150 nm) Sulfur Rhodamine 101 (Sigma Aldrich) 14.4 mg was added to 22 mL of water and dissolved, and then 2 mL of a 5% aqueous solution of Emulgen 430 (Kao) was added. After stirring at 70 ° C. while stirring on a hot stirrer, 0.65 g of melamine resin raw material Nicarax MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added. 680 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Inc.) was added and stirred with heating at 70 ° C. for 50 minutes. Thereafter, the temperature was raised to 90 ° C. and stirred for 20 minutes. In order to remove excess impurities such as resin raw materials and pigments from the obtained particle liquid, washing with pure water was performed. Centrifugation was carried out at 20000 G for 15 minutes with a centrifuge (Microcooling Centrifuge 3740 manufactured by Kubota). After removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and redispersion in ultrapure water were repeated 5 times.

  0.1 mg of the obtained particles were dispersed in 1.5 mL of EtOH, and 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and reacted for 8 hours for surface amination treatment.

  The obtained dye-encapsulated nanoparticles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. By performing washing by the same procedure three times, fluorescent dye-containing particles having a maleimide group at the end 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), then filtered through a gel filtration column, and can be bound to dye-encapsulated nanoparticles. A streptavidin solution was obtained.

  The above fluorescent nanoparticles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added 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-bound Texas Red dye-encapsulated melamine nanoparticles.

Preparation of pathological staining solution Antibody-binding fluorescent melamine resin particles (average particle size 150 nm) 0.3 nM and 7.0 g of sucrose (product name: sucrose, manufactured by Nacalai Tesque) as an additive are MilliQ water (specific resistance is 18 MΩ · cm) Water) was dispersed in 10 mL to prepare a dispersion having an additive concentration of 0.7 g / mL, which was used as a pathological staining solution.

For the pathological staining solution, the solid density ρ of the particles and the density ρ _{w of the} dispersion are measured by density gradient centrifugation using an ultracentrifuge (Hitachi, CS120FNX), and the tuning fork type vibration viscometer SV-1H (A & D) was used to measure the viscosity η.
Table 2 shows the values of solid density ρ, density ρ _w and (ρ−ρ _w ) / η of the pathological staining liquid particles.

(2) Evaluation of dispersibility After the pathological staining solution was stored at 4 ° C. for one month, the presence or absence of precipitation in the dispersion was visually evaluated.
The evaluation of dispersibility by pipetting is based on whether pipetteman (Pilpetman P-200, manufactured by Gilson Co., Ltd.) has a scale of 200 μL, sucks and discharges the pathological staining liquid, and then checks whether there is pathological staining liquid remaining on the pipette tip. This was done visually.

(3) Immunostaining method and evaluation The pipetteman scale was set to 200 μL, and the operation of sucking and discharging the pathological staining solution was repeated 5 times, and then 800 μL of PBS was added to the pathological staining solution to a concentration of 0.06 nM. The diluted pathological staining solution was poured into a centrifugal filter (manufactured by Nihon Millipore, Ultra Free MC DV 0.65 μm), and filtered by centrifuging using a desktop centrifuge.

  On the other hand, primary antibody (product name: PATHWAY anti-HER2 / neu (4B5) Rabbit Monoclonal Primary Antibody) is reacted with HER2 protein on pathological tissue slide (product name: Breast Tissue Microarray, manufactured by US Biomax). Let Subsequently, a biotin-labeled secondary antibody (product name: Universal Secondary Antibody, manufactured by Ventana Medical Systems) was reacted to form an antigen-antibody reaction conjugate on the pathological tissue slide. The combined product was stained by adding a diluted pathological staining solution.

After staining, a fluorescence image was obtained using a fluorescence microscope (manufactured by Carl Zeiss). The excitation wavelength was 575 to 600 nm, and the fluorescence wavelength was 612 to 682 nm.
Using a pathological staining liquid in which no additive was added, that is, a pathological staining liquid in which the pathological staining liquids of Comparative Examples 1-1, 2-1, 3-1, and 4-1, which will be described later, were dispersed by an ultrasonic probe. A sample subjected to fluorescent immunostaining was used as a reference.
The number of bright spots present in the tissue was visually measured from the images. When the number of bright spots was 80% or more of the reference, it was considered that the dyeing was possible. The results are shown in Table 2.

[Example 1-2] and [Example 1-3]
In Example 1-1, a pathological staining solution was prepared in the same manner as in Example 1-1 except that the concentration (g / mL) of the additive was changed to the numerical values shown in Table 2. Dispersibility (precipitation result) And weighing accuracy with a pipette) and immunostaining results were evaluated. The results are shown in Table 2.

[Comparative Example 1-1]
In Example 1-1, except that the additive was not used, a pathological staining solution was prepared in the same manner as in Example 1-1, and evaluation of dispersibility and evaluation of immunostaining were performed. The results are shown in Table 2.

[Comparative Example 1-2]
In Example 1-1, except that the concentration (g / mL) of the additive was changed to the values shown in Table 2, a pathological staining solution was prepared in the same manner as in Example 1-1, and evaluation of dispersibility and Immunostaining was evaluated. The results are shown in Table 2.

[Example 1-4]
In Example 1-1, except that glycerol was used as an additive instead of sucrose, and the additive concentration (g / mL) was changed to the values shown in Table 2, the same as Example 1-1 Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 2.

[Comparative Example 1-3] and [Comparative Example 1-4]
In Example 1-4, except that the concentration (g / mL) of the additive was changed to the numerical values shown in Table 2, a pathological staining solution was prepared in the same manner as in Example 1-4 to evaluate dispersibility and The immunostaining results were evaluated. The results are shown in Table 2.

[Example 1-5] and [Example 1-6]
In Example 1-1, except that sorbitol was used instead of sucrose as an additive, and the concentration (g / mL) of the additive was changed to the numerical values shown in Table 2, the same as Example 1-1 Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 2.

[Comparative Example 1-5]
In Example 1-5, except that the concentration (g / mL) of the additive was changed to the values shown in Table 2, a pathological staining solution was prepared in the same manner as in Example 1-5, and evaluation of dispersibility and The immunostaining results were evaluated. The results are shown in Table 2.

[Example 1-7] and [Example 1-8]
In Example 1-1, it was the same as Example 1-1 except that fructose was used instead of sucrose as an additive, and the concentration (g / mL) of the additive was changed to the numerical values shown in Table 2. Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 2.

[Comparative Example 1-6]
In Example 1-7, except that the concentration (g / mL) of the additive was changed to the values shown in Table 2, a pathological staining solution was prepared in the same manner as in Example 1-7, and evaluation of dispersibility and The immunostaining results were evaluated. The results are shown in Table 2.

[Example 1-9]
In Example 1-1, except that iopamidol was used instead of sucrose as an additive, and the concentration (g / mL) of the additive was changed to the numerical values shown in Table 2, the same as Example 1-1 Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 2.

[Comparative Example 1-7]
In Example 1-1, except that trehalose was used instead of sucrose as an additive, and the additive concentration (g / mL) was changed to the values shown in Table 2, the same as Example 1-1 Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 2.

[Comparative Example 1-8]
In Comparative Example 1-7, except that maltose was used instead of trehalose as an additive, a pathological staining solution was prepared in the same manner as Comparative Example 1-7, and evaluation of dispersibility and evaluation of immunostaining results Went. The results are shown in Table 2.

[Example 2-1]
(1) Preparation of pathological stain
Preparation of antibody-bound fluorescent melamine resin particles (average particle size 50 nm) SulfoRhodamine 101 (Sigma Aldrich) 8.1 mg was dissolved in 22 mL of water, and then 2 mL of 5% aqueous solution of Emulgen 430 (Kao Corporation) was added. After heating to 70 ° C. while stirring on a hot stirrer, 0.37 g of melamine resin raw material Nicalac MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added. 680 μL of a 10% aqueous solution of dodecyl benzene sulfonic acid (manufactured by Kanto Chemical Co., Inc.) was added and stirred with heating at 70 ° C. for 50 minutes. Thereafter, the temperature was raised to 90 ° C. and stirred for 20 minutes. In order to remove excess impurities such as resin raw materials and pigments from the obtained particle liquid, washing with pure water was performed. Centrifugation was carried out at 20000 G for 15 minutes with a centrifuge (Microcooling Centrifuge 3740 manufactured by Kubota). After removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and redispersion in ultrapure water were repeated 5 times.

  0.1 mg of the obtained particles were dispersed in 1.5 mL of EtOH, and 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and reacted for 8 hours for surface amination treatment.

  The obtained dye-encapsulated nanoparticles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. By performing washing by the same procedure three times, fluorescent dye-containing particles having a maleimide group at the end 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), then filtered through a gel filtration column, and can be bound to dye-encapsulated nanoparticles. A streptavidin solution was obtained.

  The above fluorescent nanoparticles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added 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-bound Texas Red dye-encapsulated melamine nanoparticles.

Preparation of pathological staining solution Antibody-binding fluorescent melamine resin particles (average particle size 50 nm) 0.3 nM, and 7.0 g of sucrose (product name: sucrose, manufactured by Nacalai Tesque) as an additive, MilliQ water (specific resistance 18 MΩ · cm) Water) was dispersed in 10 mL to prepare a dispersion having an additive concentration of 0.7 g / mL, which was used as a pathological staining solution.

For the pathological stain, the solid density ρ of the particles and the density ρ _{w of the} dispersion are measured by density gradient centrifugation using an ultracentrifuge (Hitachi Koki Co., Ltd., CS120FNX), and a tuning-fork vibration viscometer SV is obtained. The viscosity η was measured using -1H (manufactured by A & D).
Table 3 shows values of solid density ρ, density ρ _w and (ρ−ρ _w ) / η of the pathological staining liquid particles.

(2) Evaluation of dispersibility After the pathological staining solution was stored at 4 ° C. for one month, the presence or absence of precipitation in the dispersion was visually evaluated.
The evaluation of dispersibility by pipetting is based on whether pipetteman (Pilpetman P-200, manufactured by Gilson Co., Ltd.) has a scale of 200 μL, sucks and discharges the pathological staining liquid, and then checks whether there is pathological staining liquid remaining on the pipette tip. This was done visually.

(3) Immunostaining method and evaluation The pipetteman scale was set to 200 μL, and the operation of sucking and discharging the pathological staining solution was repeated 5 times, and then 800 μL of PBS was added to the pathological staining solution to a concentration of 0.06 nM. The diluted pathological staining solution was poured into a centrifugal filter (manufactured by Nihon Millipore, Ultra Free MC DV 0.65 μm), and filtered by centrifuging using a desktop centrifuge.

  On the other hand, primary antibody (product name: PATHWAY anti-HER2 / neu (4B5) Rabbit Monoclonal Primary Antibody) is reacted with HER2 protein on pathological tissue slide (product name: Breast Tissue Microarray, manufactured by US Biomax). Let Subsequently, a biotin-labeled secondary antibody (product name: Universal Secondary Antibody, manufactured by Ventana Medical Systems) was reacted to form an antigen-antibody reaction conjugate on the pathological tissue slide. The combined product was stained by adding a diluted pathological staining solution.

After staining, a fluorescence image was obtained using a fluorescence microscope (manufactured by Carl Zeiss).
The excitation wavelength was 575 to 600 nm, and the fluorescence wavelength was 612 to 682 nm.
The number of bright spots present in the tissue was visually measured from the images. When the number of bright spots was 80% or more of the reference, it was considered that the dyeing was possible. The results are shown in Table 3.

[Example 2-2] and [Example 2-3]
In Example 2-1, except that the concentration (g / mL) of the additive was changed to the numerical values shown in Table 3, a pathological staining solution was prepared in the same manner as in Example 2-1, and the dispersibility (precipitation result) And weighing accuracy with a pipette) and immunostaining results were evaluated. The results are shown in Table 3.

[Example 2-4]
In Example 2-1, as in Example 2-1, except that iopamidol was used instead of sucrose as an additive and the concentration (g / mL) of the additive was changed to the values shown in Table 3. Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 3.

[Comparative Example 2-1]
In Example 2-1, except that an additive was not used, a pathological staining solution was prepared in the same manner as in Example 2-1, and evaluation of dispersibility and evaluation of immunostaining were performed. The results are shown in Table 3.

[Comparative Example 2-2]
In Example 2-1, except that the concentration (g / mL) of the additive was changed to the values shown in Table 3, a pathological staining solution was prepared in the same manner as in Example 2-1, and evaluation of dispersibility and Immunostaining was evaluated. The results are shown in Table 3.

[Example 3-1]
(1) Preparation of pathological stain
Preparation of antibody-bound fluorescent melamine resin particles (average particle size 200 nm) 17.6 mg of SulfoRhodamine 101 (Sigma Aldrich) was added to 22 mL of water and dissolved, and then 2 mL of a 5% aqueous solution of Emulgen 430 (manufactured by Kao) was added. After heating to 70 ° C. with stirring on a hot stirrer, 0.80 g of melamine resin raw material Nicalac MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added. 680 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Inc.) was added and stirred with heating at 70 ° C. for 50 minutes. Thereafter, the temperature was raised to 90 ° C. and stirred for 20 minutes. In order to remove excess impurities such as resin raw materials and pigments from the obtained particle liquid, washing with pure water was performed. Centrifugation was carried out at 20000 G for 15 minutes with a centrifuge (Microcooling Centrifuge 3740 manufactured by Kubota). After removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and redispersion in ultrapure water were repeated 5 times.

  0.1 mg of the obtained particles were dispersed in 1.5 mL of EtOH, and 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and reacted for 8 hours for surface amination treatment.

  The obtained dye-encapsulated nanoparticles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. By performing washing by the same procedure three times, fluorescent dye-containing particles having a maleimide group at the end 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), then filtered through a gel filtration column, and can be bound to dye-encapsulated nanoparticles. A streptavidin solution was obtained.

  The above fluorescent nanoparticles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added 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-bound Texas Red dye-encapsulated melamine nanoparticles.

Preparation of pathological staining solution Antibody-bound fluorescent melamine resin particles 0.3 nM and 7.0 g of sucrose (product name: sucrose, manufactured by Nacalai Tesque) as an additive were dispersed in 10 mL of MilliQ water (water with a specific resistance of 18 MW · cm). Thus, a dispersion having an additive concentration of 0.7 g / mL was prepared and used as a pathological staining solution.

For the pathological stain, the solid density ρ of the particles and the density ρ _{w of the} dispersion are measured by density gradient centrifugation using an ultracentrifuge (Hitachi Koki Co., Ltd., CS120FNX), and a tuning-fork vibration viscometer SV is obtained. The viscosity η was measured using -1H (manufactured by A & D).
Table 4 shows the values of solid density ρ, density ρ _w and (ρ−ρ _w ) / η of the pathological staining liquid particles.

(2) Evaluation of dispersibility After the pathological staining solution was stored at 4 ° C. for one month, the presence or absence of precipitation in the dispersion was visually evaluated.
The evaluation of dispersibility by pipetting is based on whether pipetteman (Pilpetman P-200, manufactured by Gilson Co., Ltd.) has a scale of 200 μL, sucks and discharges the pathological staining liquid, and then checks whether there is pathological staining liquid remaining on the pipette tip. This was done visually.

(3) Immunostaining method and evaluation The pipetteman scale was set to 200 μL, and the operation of sucking up and discharging the pathological staining solution was repeated 5 times. Then, 800 μL of PBS was added to the pathological staining solution and diluted to a concentration of 0.06 nM. Then, the diluted pathological staining solution was put into a centrifugal filter (manufactured by Nihon Millipore, Ultra Free MC DV 0.65 μm), and filtered by centrifuging using a tabletop centrifuge.

  A primary antibody (product name: PATHWAY anti-HER2 / neu (4B5) Rabbit Monoclonal Primary Antibody, manufactured by Ventana Medical Systems, Inc.) is reacted with the HER2 protein on a pathological tissue slide (product name: Breast Tissue Microarray, manufactured by US Biomax). Subsequently, a biotin-labeled secondary antibody (product name: Universal Secondary Antibody, manufactured by Ventana Medical Systems) was reacted to form an antigen-antibody reaction conjugate on the pathological tissue slide. The combined product was stained by adding a diluted pathological staining solution.

After staining, a fluorescence image was obtained using a fluorescence microscope (manufactured by Carl Zeiss).
The excitation wavelength was 575 to 600 nm, and the fluorescence wavelength was 612 to 682 nm.
The number of bright spots present in the tissue was visually measured from the images. When the number of bright spots was 80% or more of the reference, it was considered that the dyeing was possible. The results are shown in Table 4.

[Example 3-2] and [Example 3-3]
In Example 3-1, a pathological staining solution was prepared in the same manner as in Example 3-1, except that the concentration (g / mL) of the additive was changed to the numerical values shown in Table 4. Dispersibility (precipitation result) And weighing accuracy with a pipette) and immunostaining results were evaluated. The results are shown in Table 4.

[Example 3-4]
In Example 3-1, the same as Example 3-1 except that iopamidol was used instead of sucrose as an additive and the concentration (g / mL) of the additive was changed to the numerical values shown in Table 4. Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 4.

[Comparative Example 3-1]
In Example 3-1, except that no additive was used, a pathological staining solution was prepared in the same manner as in Example 3-1, and evaluation of dispersibility and evaluation of immunostaining were performed. The results are shown in Table 4.

[Comparative Example 3-2]
In Example 3-1, a pathological staining solution was prepared in the same manner as in Example 3-1, except that the additive concentration (g / mL) was changed to the numerical values shown in Table 4, and evaluation of dispersibility and Immunostaining was evaluated. The results are shown in Table 4.

[Example 4-1]
(1) Preparation of pathological stain
Preparation of antibody-bound fluorescent silica particles (average particle size: 150 nm) Sulfur Rhodamine 101 (Sigma Aldrich) 14.4 mg was added to 1 ml of DMF and dissolved, and 3-aminopropyltrimethoxysilane (3-aminopropyltrimethoxysilane, Shin-Etsu Silicone, KBM903) ) 3 μL was mixed to obtain an organoalkoxysilane compound. 0.6 ml of the obtained organoalkoxysilane compound was mixed with 48 ml of ethanol, 0.6 ml of TEOS (tetraethoxysilane), 2 ml of water, 2 ml of 28% ammonia water for 3 hours. The mixed solution prepared in the above step was centrifuged at 20000 G for 15 minutes with a centrifuge (Microcooled Centrifuge 3740 manufactured by Kubota), and the supernatant was removed. After removing the supernatant, ultrapure water was added, and ultrasonic dispersion was performed to redisperse. Centrifugation, supernatant removal, and redispersion in ultrapure water were repeated 5 times.

  0.1 mg of the obtained particles were dispersed in 1.5 mL of EtOH, and 2 μL of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and reacted for 8 hours for surface amination treatment.

  The obtained dye-encapsulated nanoparticles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. By performing washing by the same procedure three times, fluorescent dye-containing particles having a maleimide group at the end 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), then filtered through a gel filtration column, and can be bound to dye-encapsulated nanoparticles. A streptavidin solution was obtained.

  The above fluorescent nanoparticles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added 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-bound Texas Red dye-encapsulated silica nanoparticles.

Preparation of pathological staining solution Antibody-bound fluorescent silica particles 0.3 nM, and 7.0 g of sucrose (product name: sucrose, manufactured by Nacalai Tesque) as an additive were dispersed in 10 mL of MilliQ water (water with a specific resistance of 18 MΩ · cm). A dispersion having an additive concentration of 0.8 g / mL was prepared as a pathological staining solution.

On the other hand, for the pathological staining solution, the solid density ρ of the particles and the density ρ _{w of the} dispersion are measured by density gradient centrifugation using an ultracentrifuge (manufactured by Hitachi Koki Co., Ltd., CS120FNX). The viscosity η was measured using a total SV-1H (manufactured by A & D).
Table 5 shows values of solid density ρ, density ρ _w and (ρ−ρ _w ) / η of the pathological staining liquid particles.

(2) Evaluation of dispersibility After the pathological staining solution was stored at 4 ° C. for one month, the presence or absence of precipitation in the dispersion was visually evaluated.
The evaluation of dispersibility by pipetting is based on whether pipetteman (Pilpetman P-200, manufactured by Gilson Co., Ltd.) has a scale of 200 μL, sucks and discharges the pathological staining liquid, and then checks whether there is pathological staining liquid remaining on the pipette tip. This was done visually.

(3) Immunostaining method and evaluation The pipetteman scale was set to 200 μL, and the operation of sucking and discharging the pathological staining solution was repeated 5 times, and then 800 μL of PBS was added to the pathological staining solution to a concentration of 0.06 nM. The diluted pathological staining solution was poured into a centrifugal filter (manufactured by Nihon Millipore, Ultra Free MC DV 0.65 μm), and filtered by centrifuging using a desktop centrifuge.

  A primary antibody (product name: PATHWAY anti-HER2 / neu (4B5) Rabbit Monoclonal Primary Antibody) manufactured by Ventana Medical Systems is reacted with the HER2 protein on a pathological tissue slide (product name: Breast Tissue Microarray, product of US Biomax). Subsequently, a biotin-labeled secondary antibody (product name: Universal Secondary Antibody, manufactured by Ventana Medical Systems) was reacted to form an antigen-antibody reaction conjugate on the pathological tissue slide. The combined product was stained by adding a diluted pathological staining solution.

After staining, a fluorescence image was obtained using a fluorescence microscope (manufactured by Carl Zeiss).
The excitation wavelength was 575 to 600 nm, and the fluorescence wavelength was 612 to 682 nm.
The number of bright spots present in the tissue was visually measured from the images. When the number of bright spots was 80% or more of the reference, it was considered that the dyeing was possible. The results are shown in Table 5.

[Example 4-2]
In Example 4-1, except that the concentration (g / mL) of the additive was changed to the values shown in Table 5, a pathological staining solution was prepared in the same manner as in Example 4-1, and the dispersibility (precipitation result) And weighing accuracy with a pipette) and immunostaining results were evaluated. The results are shown in Table 5.

[Comparative Example 4-1]
In Example 4-1, except that the additive was not used, a pathological staining solution was prepared in the same manner as in Example 4-1, and evaluation of dispersibility and evaluation of immunostaining were performed. The results are shown in Table 5.

[Comparative Example 4-2] and [Comparative Example 4-3]
In Example 4-1, except that the concentration (g / mL) of the additive was changed to the numerical values shown in Table 5, a pathological staining solution was prepared in the same manner as in Example 4-1, and evaluation of dispersibility and The immunostaining results were evaluated. The results are shown in Table 5.

[Comparative Example 4-4]
In Example 4-1, similar to Example 4-1, except that iopamidol was used instead of sucrose as an additive and that the concentration (g / mL) of the additive was changed to the numerical values shown in Table 5. Thus, a pathological staining solution was prepared, and the dispersibility was evaluated and the immunostaining result was evaluated. The results are shown in Table 5.

[Preparation Example 1] Preparation of antibody-bound fluorescent melamine resin particles 14.4 mg of Sulfur Rhodamine 101 (manufactured by Sigma Aldrich) as a fluorescent dye was added to 22 mL of water and dissolved. Thereafter, 2 mL of a 5% aqueous solution of Emulgen (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation), an emulsifier for emulsion polymerization, was added to this solution. This solution was heated to 70 ° C. while stirring on a hot stirrer, and then 0.65 g of melamine resin raw material Nicalak MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added to this solution. Further, 1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Inc.) as a reaction catalyst and surfactant was added to this solution, and the mixture was heated and stirred at 70 ° C. for 50 minutes. Then, it heated up at 90 degreeC and heat-stirred for 20 minutes. In order to remove impurities such as excess resin raw materials and fluorescent dyes from the obtained dispersion of the dye resin particles, washing with pure water was performed.

  Specifically, the mixture was centrifuged at 20000 G for 15 minutes in a centrifuge (Kubota Micro Cooling Centrifuge 3740), and after removing the supernatant, ultrapure water was added and ultrasonically irradiated to redisperse. Centrifugation, supernatant removal, and washing by redispersion in ultrapure water were repeated 5 times. The obtained melamine particles were positively charged because the melamine resin itself contains many amino groups in the skeleton. The charge of the resin particles was evaluated by resin composition analysis by IR or the like and zeta potential measurement.

  Mixing 1 mL of the washed 0.67 nM aqueous dispersion of melamine resin particles with 20 mg of 1,2-Bis (2-aminoethoxy) ethane and reacting at a temperature of 70 ° C. for 20 minutes to convert the surface into amino groups I did it. That is, amino groups of 1,2-Bis (2-aminoethoxy) ethane were reacted with hydroxyl groups of melamine resin particles to introduce amino groups. The obtained melamine resin particles were repeatedly washed three times by removing the supernatant by centrifugation and redispersing in ultrapure water.

  The melamine particles into which amino groups were introduced were dispersed in THF, then precipitated by centrifugation, and dehydrated by being dispersed again in THF. The particle concentration at that time was 0.67 nM. Thereafter, 3 mg of SM (PEG) 12 (Succinimidyl-[(N-maleimidepropionamid) -dodecaethyleneglycol) ester, manufactured by Thermo Scientific Co., Ltd.) was mixed with the dispersion of the dye resin particles whose concentration was adjusted, and 20 ° C. for 1 hour. By reacting, a mixed liquid containing dye resin particles having a fluorescent dye with a maleimide at the end was obtained.

  The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM EDTA was added to disperse the precipitate, and the mixture was centrifuged again. The above washing according to the same procedure was performed three times.

(Preparation of streptavidin)
On the other hand, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) and 2-Iminothiolane · HCl (abbreviation: Traut's reagent) are used to add a thiol group to streptavidin, and gel filtration is performed on the dye resin particles. A streptavidin capable of binding was prepared separately.

(Binding of resin particles and streptavidin)
The dye resin particles and streptavidin were mixed in PB containing 2 mM EDTA and then reacted at room temperature for 1 hour to carry out a reaction for bonding them. After the reaction, 10 mM mercaptoethanol was added to stop the reaction. The obtained solution was concentrated with a centrifugal filter of φ = 0.65 μm, and then unreacted streptavidin and the like were removed using a purification gel filtration column to obtain dye resin particles bound with streptavidin.

[Example 5-1]
Preparation of aqueous dispersion 0.5 mg of xanthan gum as a thixotropic agent was added to 1 mL of 1% BSA (manufactured by Dako) / PBS dispersion containing 0.3 nM antibody-bound fluorescent melamine resin particles, and the concentration of xanthan gum was 0.5 A mg / mL aqueous dispersion was prepared. As BSA, BSA (bovine serum albumin) manufactured by Dako was used.
Evaluation of stability after storage of aqueous dispersion for one week (hereinafter referred to as “Evaluation 1”)

  The aqueous dispersion was put into a reagent bottle of an automatic staining apparatus (manufactured by Ventana, XT System Discovery), and 150 μL was discharged 5 times, and the standard deviation was calculated from the concentration of each particle. After one week with the aqueous dispersion in the reagent bottle, 150 μL was again discharged five times, and the standard deviation was calculated from the concentration of each particle. The particle concentration was calculated by setting the luminance with a fluorometer (F-7000, manufactured by Hitachi, Ltd.) and setting the luminance of the same amount of dispersion before discharge as 100.

The smaller the standard deviation, the smaller the dispersion of particles in the aqueous dispersion. The standard deviation when measured immediately after adding the aqueous dispersion and the standard deviation measured after one week has passed. The smaller the difference, the better the storage stability.
The results are shown below.
Standard deviation (measured immediately after input) 5
Standard deviation (after one week) 8

Measurement of apparent viscosity After leaving overnight 10 mL of aqueous dispersion, stir with a magnetic stirrer for about 5 minutes to make a complete solution, transfer to a container with a lid of about 45 mm in diameter and about 145 mm in height, 25 ± 0.2 After standing for 30 minutes in a thermostat at ℃, gently stir the solution with a glass rod, attach the rotor (No.1) and guard of B type viscometer (BII viscometer manufactured by Toki Sangyo Co., Ltd.) Rotate and read scale after 3 minutes.
Viscosity (mPa · s) = reading scale × coefficient The apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C. is 45 mPa.s. s.

[Example 5-2]
In Example 5-1, the concentration of CMC-Na was 20 mg / ml in the same manner as in Example 5-1, except that carboxymethyl cellulose sodium salt (CMC-Na) was used instead of xanthan gum as the thixotropic agent. mL aqueous dispersion was prepared.
The results of evaluation 1 using the above aqueous dispersion are shown below.
Standard deviation (measured immediately after input) 4
Standard deviation (after standing for one week) 5

Measurement of apparent viscosity The apparent viscosity was measured under the same conditions as in Example 5-1, and the apparent viscosity (B-type viscometer, 70 rpm) of the aqueous dispersion at 25 ° C was 56 mPa.s. s.

[Example 5-3]
In Example 5-1, an aqueous dispersion having a guar gum concentration of 1 mg / mL was prepared in the same manner as in Example 5-1, except that guar gum was used instead of xanthan gum as the thixotropic agent.
The results of evaluation 1 using the above aqueous dispersion are shown below.
Standard deviation (measured immediately after input) 6
Standard deviation (after standing for one week) 8

Measurement of apparent viscosity The apparent viscosity was measured under the same conditions as in Example 5-1, and the apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C was 72 mPa.s. s.

[Example 5-4]
In Example 5-1, as the thixotropy-imparting agent, Emanon 3299V (Kao Corporation), which is a PEG nonionic thickener, was used instead of xanthan gum. An aqueous dispersion having a concentration of 3299 V of 10 mg / mL was prepared.
The results of evaluation 1 using the above aqueous dispersion are shown below.
Standard deviation (measured immediately after input) 5
Standard deviation (after standing for one week) 6

Measurement of apparent viscosity The apparent viscosity was measured under the same conditions as in Example 5-1, and the apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C was 80 mPa.s. s.

[Comparative Example 5-1]
In Example 5-1, an aqueous dispersion was prepared in the same manner as in Example 5-1, except that the thixotropic agent was not used.
The results of evaluation 1 using the above aqueous dispersion are shown below.
Standard deviation (measured immediately after input) 13
Standard deviation (after standing for one week) 85

Measurement of apparent viscosity The apparent viscosity was measured under the same conditions as in Example 5-1, and the apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C was 1.8 mPa.s. s.

[Comparative Example 5-2]
In Example 5-1, an aqueous dispersion having a sucrose concentration of 750 mg / mL was prepared in the same manner as in Example 5-1, except that sucrose was used instead of the thixotropic agent.
The results of evaluation 1 using the above aqueous dispersion are shown below.
Standard deviation (measured immediately after input) 8
Standard deviation (after standing for one week) 52

Measurement of apparent viscosity The apparent viscosity was measured under the same conditions as in Example 5-1, and the apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C was 50 mPa.s. s.

[Comparative Example 5-3]
In Example 5-1, an aqueous dispersion having a glycerol concentration of 950 mg / mL was prepared in the same manner as in Example 5-1, except that glycerol was used instead of the thixotropic agent.
The results of evaluation 1 using the above aqueous dispersion are shown below.
Standard deviation (measured immediately after input) 11
Standard deviation (after standing for one week)-(not measurable)

  In Comparative Examples 5-1 to 5-3, compared with Examples 5-1 to 5-3, both when the water dispersion was measured immediately after measurement and when measured after standing for one week The standard deviation value is large, and the difference between the standard deviation when measured immediately after adding the aqueous dispersion and the standard deviation measured after standing for one week (how to increase the dispersion) is large. I understand.

  Therefore, in Examples 5-1 to 5-3, compared with Comparative Examples 5-1 to 5-3, by adding the thixotropy-imparting agent, the dispersion of the particles in the aqueous dispersion is small, and the mixture is allowed to stand for one week. It turns out that it is excellent also in the storage stability after.

Measurement of apparent viscosity The apparent viscosity was measured under the same conditions as in Example 5-1, and the apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C was 40 mPa.s. s.

[Example 6-1]
Preparation of aqueous dispersion 1.0 mg of xanthan gum as a thixotropic agent was added to 1 mL of 1% BSA / PBS dispersion containing antibody-bound fluorescent melamine resin particles at a concentration of 0.3 nM, and water with xanthan gum concentration of 1.0 mg / mL A dispersion was prepared.

Measurement of apparent viscosity After leaving overnight 10 mL of aqueous dispersion, stir with a magnetic stirrer for about 5 minutes to make a complete solution, transfer to a container with a lid of about 45 mm in diameter and about 145 mm in height, 25 ± 0.2 After standing for 30 minutes in a thermostat at ℃, gently stir the solution with a glass rod, attach the rotor (No.1) and guard of B type viscometer (BII viscometer manufactured by Toki Sangyo Co., Ltd.) Rotate and read scale after 3 minutes.
Viscosity (mPa · s) = reading scale × coefficient The apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C. is 80 mPa.s. s.

Evaluation of dispersibility of particles on APS-coated glass slide (hereinafter referred to as “Evaluation 2”)
Aqueous dispersion is put into a reagent bottle of an automatic staining apparatus (manufactured by Ventana Co., Ltd., XT System Discovery), and 150 μL each is continuously discharged onto five APS (aminosilane) coated slide glasses (manufactured by Matsunami Glass Industrial Co., Ltd.) The number of bright spots on each APS-coated slide glass was measured with a fluorescence microscope (manufactured by Olympus, BX53), and the standard deviation was calculated from the number of bright spots.

The larger the number of bright spots, the more the particles are not aggregated or precipitated in the aqueous dispersion and the better dispersibility is maintained. Therefore, the higher the average value of the number of bright spots and the smaller the standard deviation, the better the dispersibility of the number of bright spots on the APS-coated slide glass.
The average value and standard deviation of the number of bright spots carried out 5 times are shown below.
Number of bright spots (average) 2930
Standard deviation 14

[Example 6-2]
In Example 6-1, Example 6-1, except that 0.5 mg of xanthan gum was added instead of 1.0 mg as a thixotropy imparting agent, and the concentration of xanthan gum was set to 0.5 mg / mL instead of 1.0 mg / mL. Similarly, a dispersion was prepared.

The apparent viscosity (B-type viscometer, 60 rpm) at 25 ° C. of the aqueous dispersion was 45 mPa.s. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average value) 4632
Standard deviation 7

[Example 6-3]
In Example 6-1, a dispersion was prepared in the same manner as in Example 6-1, except that carboxymethyl cellulose sodium salt (CMC-Na) was used instead of xanthan gum as the thixotropy-imparting agent. The concentration was 22 mg / mL, which was the same as in Example 6-1.

The apparent viscosity (B-type viscometer, 60 rpm) at 25 ° C. of the aqueous dispersion was 70 mPa.s. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average) 2239
Standard deviation 12

[Example 6-4]
In Example 6-3, a dispersion was prepared in the same manner as in Example 6-3 except that the concentration of carboxymethylcellulose sodium salt (CMC-Na) was 20 mg / mL instead of 1.0 mg / mL. .

The apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C. is 56 mPa.s. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average value) 3150
Standard deviation 11

[Example 6-5]
In Example 6-3, a dispersion was prepared in the same manner as in Example 6-3, except that the concentration of carboxymethylcellulose sodium salt (CMC-Na) was 16 mg / mL instead of 1.0 mg / mL. .

The apparent viscosity (B-type viscometer, 60 rpm) at 25 ° C. of the aqueous dispersion was 30 mPa.s. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average value) 5212
Standard deviation 4

[Example 6-6]
In Example 6-1, a dispersion was prepared in the same manner as in Example 6-1, except that guar gum was used instead of xanthan gum as the thixotropy-imparting agent. The concentration was 1.0 mg / mL, which was the same as in Example 6-1.

The apparent viscosity (B-type viscometer, 60 rpm) at 25 ° C. of the aqueous dispersion was 72 mPa.s. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average value) 3042
Standard deviation 10

[Example 6-7]
In Example 6-1, as a thixotropy-imparting agent, a dispersion liquid was used in the same manner as in Example 6-1, except that Emanon 3299V (Kao Corporation), which is a PEG nonionic thickener, was used instead of xanthan gum. Was prepared. The concentration was also 1.2 mg / mL, the same as in Example 6-1.

The apparent viscosity (B-type viscometer, 60 rpm) at 25 ° C. of the aqueous dispersion was 80 mPa.s. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average value) 3120
Standard deviation 12

[Comparative Example 6-1]
In Example 6-1, a dispersion was prepared in the same manner as in Example 6-1, except that the thixotropic agent was not used.

The viscosity of the aqueous dispersion was measured with a B-type viscometer (BII-type viscometer manufactured by Toki Sangyo Co., Ltd.) and 1.8 mPa. s.
The results of evaluation 2 using the above aqueous dispersion are shown below.
Number of bright spots (average value) 750
Standard deviation 15

  In Comparative Example 6-1, the viscosity is low and the average number of bright spots is small compared to Examples 6-1 to 6-5. This is because the particles are aggregated or precipitated in the dispersion due to low viscosity, causing clogging during discharge, and the aqueous dispersion could not be placed on the APS-coated slide glass in an accurate amount.

  That is, in Examples 6-1 to 6-5, it can be seen that the dispersibility of particles on the APS-coated slide glass is improved by adding the thixotropy imparting agent as compared with Comparative Example 6-1.

[Reference Example 6-1]
Here, as a reference, for xanthan gum aqueous solutions having concentrations of 0.05%, 0.25%, 0.50% and 1.00%, a B-type viscometer (BII viscometer manufactured by Toki Sangyo Co., Ltd.) and a rotor Table 6 shows the relationship between the rotational speed of the rotor and the apparent viscosity when the rotational speed of the rotor (NO.1) is 6 rpm, 12 rpm, 30 rpm, and 60 rpm using (NO.1).

From Table 6, it can be seen that the apparent viscosity decreases as the rotational speed of the rotor of the viscometer increases, regardless of the concentration of the xanthan gum aqueous solution.

[Example 7-1]
Preparation of aqueous dispersion 20 mg of carboxymethylcellulose sodium salt (CMC-Na) as a thixotropic agent was added to 1 mL of 1% BSA / PBS dispersion containing 0.3 nM of antibody-bound fluorescent melamine resin particles, An aqueous dispersion having a salt (CMC-Na) concentration of 20 mg / mL was prepared. Acetic acid was added to this aqueous dispersion, and the pH was adjusted to 5 using a pH meter (manufactured by Horiba, Ltd.).

The apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C. is 56 mPa.s. s.
Evaluation of immunohistochemical staining under a predetermined pH condition (hereinafter referred to as “Evaluation 3”)
An aqueous dispersion is put into a reagent bottle of an automatic staining apparatus (manufactured by Ventana, XT System Discovery), 150 μL is discharged onto an APS-coated slide glass on which a tissue section is placed, and the number of bright spots on the APS-coated slide glass is measured with a fluorescence microscope ( It measured by Olympus BX53) and confirmed visually.
The results are shown below.
Number of bright spots 2342

[Example 7-2]
A dispersion was prepared in the same manner as in Example 7-1 except that the pH was changed to 9 instead of 5 in Example 7-1, and Evaluation 3 was performed.
The results are shown below.
Number of bright spots 2845

[Example 7-3]
In Example 7-1, a dispersion was prepared and evaluated 3 in the same manner as in Example 7-1 except that the pH was set to 7 instead of 5.
The results are shown below.
Number of bright spots 4532

  In Example 7-3, since the number of bright spots is observed more than in Examples 7-1 and 7-2, it can be seen that the closer the pH is to neutral, the better the dyeability. A possible cause of this is that when the pH deviates from neutrality, the avidin-biotin reaction becomes difficult to proceed.

[Reference Example 7-1]
In Example 7-3, the thixotropic agent was not used, and the apparent viscosity (B-type viscometer, 60 rpm) of the aqueous dispersion at 25 ° C. was 56 mPa.s. s, not 1.8 mPa.s. A dispersion was prepared and evaluated 3 in the same manner as in Example 3-3 except that s was set.
The results are shown below.
Number of bright spots 532

Claims (6)

A pathological staining solution comprising a dispersion satisfying the following formula (1), comprising a fluorescent label comprising fluorescent nanoparticles having an average particle diameter of 50 to 200 nm and a solvent.
(Ρ−ρ _w ) /η≦0.02 (1)
(In the formula (1), ρ represents the solid density (g / cm ³ ) of the fluorescent nanoparticles at 20 ° C., ρ _w represents the density of the dispersion (g / cm ³ ) at 20 ° C., and η is 20 ° C. Represents the viscosity (g / cm · sec) of the dispersion liquid in which 1 <η <100 .) The pathological staining solution according to claim 1 , further comprising a monosaccharide, a disaccharide or an X-ray contrast agent as an additive for adjusting density and / or viscosity in addition to the fluorescent label and the solvent. The pathological stain according to claim 2 , wherein the additive for adjusting density and / or viscosity is at least one selected from the group consisting of sucrose, glycerol, sorbitol, fructose, and iopamidol.   The pathological stain according to claim 1, wherein the dispersion is an aqueous dispersion containing a fluorescent label, a thixotropy imparting agent, and an aqueous solvent for dispersing or dissolving these two components. The pathological staining solution according to claim 4 , wherein the thixotropic agent is a water-soluble polymer. The density of the said dispersion liquid is 1.00-3.00g / cm < ³ >, The pathological stain liquid as described in any one of Claims 1-5.