DILUENT FOR FLUORESCENT NANO PARTICLES, KIT FOR IMMUNOFLUORESCENT STAINING WHICH UTILIZES SAME, SOLUTION FOR IMMUNOFLUORESCENT STAINING, IMMUNOFLUORESCENT STAINING METHOD, AND GENE STAINING METHOD

Abstract

Provided is a means for detecting and quantifying a biological substance of interest with an improved accuracy by inhibiting non-specific adsorption of fluorescent nanoparticles and thereby reducing the background noise in immunostaining with fluorescent nanoparticles. Immunostaining is carried out upon diluting fluorescent nanoparticles with a fluorescent nanoparticle diluent which contains 1 to 5% (W/W) of a protein having a molecular weight of 40,000 or higher (e.g., BSA) and 1 to 3% (W/W) of a protein having a molecular weight of less than 40,000 (e.g., casein) and, when casein is used as a low-molecular-weight protein, it is preferred that the -casein content in the casein is 10% (W/W) or less and the ratio of -casein and -casein (-casein:-casein) contained in the casein is 40:60 to 60:40 (taking the total amount of -casein and -casein as 100).

Claims

1.-18. (canceled)

19. An immunofluorescent staining method, comprising: a step of immunostaining a tissue section using a fluorescent particle dispersed in a diluent comprising 1 to 5% (W/W) of a protein having a molecular weight of 40,000 or higher and 1 to 3% (W/W) of a protein having a molecular weight of less than 40,000.

20. The immunofluorescent staining method according to claim 19, wherein the fluorescent particle binds directly or indirectly to a substance of interest in the tissue section.

21. The immunofluorescent staining method according to claim 19, wherein the fluorescent particle is a nano-sized particle.

22. The immunofluorescent staining method according to claim 19, wherein the fluorescent particle comprises an organic or inorganic matrix substance and a plurality of fluorescent substances.

23. The immunofluorescent staining method according to claim 19, wherein said protein having a molecular weight of 40,000 or higher is BSA.

24. The immunofluorescent staining method according to claim 19, wherein said protein having a molecular weight of less than 40,000 is casein.

25. The immunofluorescent staining method according to claim 24, wherein a ratio of -casein in said casein is 0 to 10% (W/W).

26. The immunofluorescent staining method according to claim 24, wherein a ratio of -casein to -casein (-casein:-casein) contained in said casein is 40:60 to 60:40 (taking the total amount of -casein and -casein as 100).

Description

EXAMPLES

Preparation Example 1

Preparation of Biotin-Modified Anti-Rabbit IgG Antibody

[0108] In a 50 mM Tris solution, 50 g of an anti-rabbit IgG antibody to be used as a secondary antibody was dissolved. To the resulting solution, a DTT (dithiothreitol) solution was added to a final concentration of 3 mM, and the resultant was mixed and allowed to react at 37 C. for 30 minutes. Then, the thus obtained reaction solution was passed through a desalting column Zeba Desalt Spin Columns (manufactured by Thermo Fisher Scientific K.K., Cat.# 89882) to purify a DTT-reduced secondary antibody. An antibody solution was prepared by dissolving 200 L of the whole amount of the thus purified antibody in a 50 mM Tris solution. Meanwhile, a linker reagent Maleimide-PEG2-Biotin (manufactured by Thermo Fisher Scientific K.K., Product No. 21901) was adjusted with DMSO to a concentration of 0.4 mM. Then, 8.5 L of this linker reagent solution was added to the antibody solution, and the resultant was mixed and allowed to react at 37 C. for 30 minutes, whereby biotin was bound to the anti-rabbit IgG antibody via a PEG chain. The resulting reaction solution was purified through a desalting column. The absorbance of the thus desalted reaction solution was measured at a wavelength of 300 nm using a spectrophotometer (F-7000, manufactured by Hitachi, Ltd.) to determine the amount of the protein (biotin-modified secondary antibody) contained in the reaction solution. The reaction solution was adjusted with a 50 mM Tris solution to have a biotin-modified secondary antibody concentration of 250 g/mL, and the resulting solution was used as a biotin-modified secondary antibody solution (reagent II).

Preparation Example 2

Preparation of Texas Red Dye-Containing Melamine Resin Nanoparticles

[0109] After dissolving 2.5 mg of Texas Red dye molecule Sulforhodamine 101 (manufactured by Sigma-Aldrich) in 22.5 mL of pure water, the resulting solution was stirred for 20 minutes using a hot stirrer with the temperature of the solution being maintained at 70 C. Then, 1.5 g of a melamine resin Nikalac MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added to the solution, and the resultant was further stirred with heating for 5 minutes under the same conditions. To the thus heat-stirred solution, 100 L of formic acid was added, and the resulting solution was stirred for 20 minutes with its temperature being maintained at 60 C., after which the solution was left to stand and allowed to cool to room temperature. The thus cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to allow the Texas Red dye-containing melamine resin nanoparticles contained as a mixture in the solution to precipitate. After removing the resulting supernatant, the precipitated particles were washed with ethanol and water. Thereafter, 1,000 of the thus obtained nanoparticles were observed under an SEM and their average particle size was measured as described above, as a result of which the average particle size was found to be 152 nm.

Preparation Example 3

Preparation of Streptavidin-Modified Texas Red Dye-Containing Melamine Resin Nanoparticles

[0110] First, 0.1 mg of the particles obtained in Preparation Example 2 was dispersed in 1.5 mL of ethanol, and 2 L of aminopropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added thereto. The resulting mixture was allowed to react for 8 hours to perform a surface amination treatment.

[0111] Then, the thus surface-aminated particles were adjusted with PBS (phosphate-buffered physiological saline) containing 2 mM of EDTA (ethylenediamine tetraacetic acid) to a concentration of 3 nM, and this solution was mixed with SM(PEG)12 (succinimidyl-[(N-maleimidopropionamid)-dodecaethylene glycol]ester, manufactured by Thermo Fisher Scientific K.K.) to a final concentration of 10 mM and allowed to react for 1 hour. This mixture was centrifuged at 10,000 G for 20 minutes and the resulting supernatant was removed, after which PBS containing 2 mM of EDTA was added to disperse the precipitates, and the resulting dispersion was centrifuged again. The precipitates were washed three times by the same procedure to obtain fluorescent substance-containing melamine nanoparticle having a maleimide group at a terminal.

[0112] Meanwhile, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) was subjected to a thiol group addition treatment with N-succinimidyl-S-acetylthioacetate (SATA) and subsequently filtered through a gel-filtration column to obtain a solution of streptavidin capable of binding to the fluorescent substance-containing melamine nanoparticles.

[0113] The thus obtained fluorescent substance-containing melamine nanoparticles and streptavidin were mixed in PBS containing 2 mM of EDTA and allowed to react for 1 hour at room temperature. Then, the reaction was terminated with an addition of 10 mM mercaptoethanol. After concentrating the resulting solution using a centrifugation filter, unreacted streptavidin and the like were removed using a purification gel-filtration column, whereby fluorescent substance-containing melamine nanoparticles bound with streptavidin were obtained.

Preparation Example 4

Preparation of Melamine Resin Particles Containing CdSe/ZnS Semiconductor Nanoparticles Having Carboxylate Group as Surface-Modifying Group

[0114] Under an argon flow, 2.9 g of stearic acid, 620 mg of n-tetradecyl phosphonic acid and 250 mg of cadmium oxide were added to 7.5 g of tri-n-octylphosphine oxide, and the resultant was heated to 370 C. and mixed. After cooling the resulting mixture to 270 C., a solution prepared by dissolving 200 mg of selenium in 2.5 mL of tributyl phosphine was added thereto, and the resultant was dried under reduced pressure to obtain cadmium selenide (CdSe)-core semiconductor nanoparticles coated with tri-n-octylphosphine oxide.

[0115] Then, 15 g of tri-n-octylphosphine oxide was added to the thus obtained CdSe-core semiconductor nanoparticles and the resultant was heated, after which a solution prepared by dissolving 1.1 g of zinc diethyldithiocarbamate in 10 mL of trioctylphosphine was added thereto at 270 C., whereby a CdSe/ZnS semiconductor nanoparticle-containing dispersion was obtained.

[0116] The thus obtained dispersion was added to decane such that the CdSe/ZnS semiconductor nanoparticles were dispersed at a concentration of 5% by mass. Surface modification was performed by adding 0.5 mL of sodium propionate to 10 L of the resulting dispersion and stirring the resultant at room temperature. After adding 2.5 mL of pure water to this reaction mixture, the resulting solution was stirred for 20 minutes using a hot stirrer with the temperature of the solution being maintained at 70 C. Then, 1.5 g of a melamine resin Nikalac MX-035 (manufactured by Nippon Carbide Industries Co., Ltd.) was added to the solution, and the resultant was further stirred with heating for 5 minutes under the same conditions.

[0117] To the thus stirred solution, 100 L of formic acid was added, and the resulting solution was stirred for 20 minutes with its temperature being maintained at 60 C., after which the solution was left to stand and allowed to cool to room temperature. The thus cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to allow the melamine resin nanop articles contained as a mixture in the solution to precipitate. The resulting supernatant was removed, and the precipitated particles were subsequently washed with ethanol and water, whereby nanoparticles (quantum dot-containing melamine resin nanoparticles) having an average particle size of 150 nm were prepared.

Preparation Example 5

Preparation of Streptavidin-Modified Quantum Dot-Containing Melamine Resin Nanoparticles

[0118] Streptavidin-modified quantum dot-containing melamine resin nanoparticles were obtained from 0.1 mg of the nanoparticles obtained in Preparation Example 4 in the same manner as in Preparation Example 3.

Experimental Example 1

(E1) Sample Preparation Step

[0119] A breast cancer tissue array slide (tissue section-mounted glass slide; br243, manufactured by US Biomax, Inc.) was purchased and, using Ventana I-VIEW PATHWAY HER2 (4B5) kit, the tissue array slide was stained with Ventana BenchMark ULTRA, and the HER2 3+ region (i.e., cancer cell region) and the interstitial cell region were morphologically identified by a DAB method.

[0120] This sample was deparaffinized and then washed for replacement with water. The thus washed tissue array slide was subjected to a 15-minute autoclave treatment at 121 C. in 10 mM citrate buffer (pH 6.0), thereby performing an antigen retrieval treatment. After the retrieval treatment, the tissue array slide was washed with PBS and then subjected to a 1-hour blocking treatment with 1% BSA-containing PBS.

(E2) Immunostaining Step

(E2-1) Primary Reaction Treatment of Immunostaining

[0121] As a primary reaction treatment for the first immunostaining of a biological substance of interest HER2, a primary reaction treatment liquid containing an anti-HER2 rabbit monoclonal antibody 4B5 (manufactured by Ventana Medical Systems, Inc.) at a concentration of 0.05 nM was prepared using 1-W/W% BSA-containing PBS, and the sample prepared in the step (1) was immersed in this primary reaction treatment liquid and allowed to react overnight at 4 C.

(E2-2) Secondary Reaction Treatment of Immunostaining

[0122] A secondary reaction treatment liquid was prepared by further diluting the biotin-modified anti-rabbit IgG antibody solution prepared in Preparation Example 1 with 1-W/W% BSA-containing PBS to a concentration of 6 g/mL. The sample subjected to the primary reaction treatment was washed with PBS and subsequently immersed in this secondary reaction treatment liquid and allowed to react at room temperature for 30 minutes.

(E2-3) Fluorescent Labeling Treatment of Immunostaining

[0123] Fluorescent labeling reaction treatment liquids were each prepared by diluting the streptavidin-modified Texas Red dye-containing melamine resin particles prepared in Preparation Example 3 to a concentration of 0.02 nM with the respective fluorescent nanoparticle diluents having different content ratios of caseins (composition, -casein (c6780, manufactured by Sigma-Aldrich): 50 W/W%, -casein (c6905, manufactured by Sigma-Aldrich): 50 W/W%) and BSA as shown in Table 1. The sample subjected to the secondary reaction treatment was immersed in these fluorescent labeling treatment liquids and allowed to react at room temperature for 3 hours under neutral pH environment (pH 6.9 to 7.4).

(E3) Sample Post-Treatment Step

[0124] The thus immunostained sample was subjected to a fixation-dehydration treatment where the sample was immersed in pure ethanol for 5 minutes four times. Subsequently, the sample was subjected to a clearing treatment where the sample was immersed in xylene for 5 minutes four times. Finally, the sample was subjected to a mounting treatment where a mounting medium Entellan New (manufactured by Merck KGaA) was placed on the sample and a cover glass was further set thereon, whereby a sample for observation was prepared.

(E4) Evaluation Step

(E4-1) Observation and Image-Capturing Step

[0125] In this step, a fluorescence microscope BX-53 (manufactured by Olympus Corporation) was used for irradiation of an excitation light and observation of emitted fluorescence, and a microscope digital camera DP73 (manufactured by Olympus Corporation) attached to the fluorescence microscope was used for taking immunostained images (400).

[0126] First, the sample was irradiated with an excitation light to cause the Texas Red dye, which was used for the fluorescent labeling of the biological substance of interest HER2, to emit fluorescence, and an immunostained image in this state was photographed. In this process, the wavelength of the excitation light was set at 575 to 600 nm using an excitation light optical filter installed in the fluorescence microscope, and the wavelength of the fluorescence to be observed was set at 612 to 692 nm using a fluorescence optical filter. The intensity of the excitation light in the observation and image capturing under the fluorescence microscope was set such that an irradiation energy of 900 W/cm.sup.2 was provided in the vicinity of the center of the visual field. The exposure time for the image capturing was adjusted in such a range that does not cause saturation of the image brightness, and it was set, for example, at 4,000 sec.

[0127] After immunostained images were captured in a single visual field, the same operations were repeated in different visual fields to capture immunostained images in a total of five visual fields (first to fifth visual fields) for each sample.

(E4-2) Image Processing and Measurement Step

[0128] For the image processing in this step, an image processing software ImageJ (open source) was used.

[0129] In each immunostained image, among the bright sports representing the Texas Red dye-containing melamine resin particles with which HER2 was fluorescently labeled, ones having a brightness of not less than a prescribed value were counted.

[0130] The interstitial noise, the number of bright spots per cell of the HER2 3+ region (that is, cancer cell region) and the number of aggregated bright spots were measured. The results thereof are shown in Table 1. It is noted here that, since HER2 is not expressed in the interstitial cell region, those bright spots positioned inside the interstitial cells are non-specific signals, that is, noise. A large number of bright spots representing the interstitial noise means the occurrence of non-specific reactions in a large number; therefore, the number of such bright spots was used as an evaluation index of immunoreaction.

(In Table 1 above, % means W/W%. The same applies to Tables 2 to 4 below.)

[0131] A=Interstitial noise (Number of bright spots (the same applies below)) [0132] 500 or more=x [0133] 300 to less than 500= [0134] 200 to less than 300= [0135] Less than 200=

[0136] B=Number of bright spots per cell [0137] Less than 10=x [0138] 10 or more=

[0139] C=Aggregated bright spots [0140] 3 or more=x [0141] 3 or less=

Experimental Example 2

[0142] The interstitial noise, the number of bright spots per cell and the number of aggregated bright spots were measured by performing the sample preparation step, the immunostaining step and the evaluation step in the same manner as in Experimental Example 1, except that the composition of caseins contained in the solution used for dispersing the fluorescent nanoparticles in (E2-3) Fluorescent Labeling Treatment of Immunostaining was changed. The results thereof are shown in Tables 2 to 4. It is noted here that the content ratios of the respective caseins in the natural casein are: -casein:50%, -casein:35%, -casein:13%, and -casein:2%.

TABLE-US-00001 TABLE 2 Casein (natural) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: 300 to less than 500 = 3.0% Number of bright spots per cell: 10 or more = 5.0% Aggregated bright spot: none =

TABLE-US-00002 TABLE 3 Casein (-casein: 45%, -casein: 45%, -casein: 10%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: less than 200 = 3.0% Number of bright spots per cell: 10 or more = 5.0% Aggregated bright spot: none =

TABLE-US-00003 TABLE 3A Casein (-casein: 49.5%, -casein: 49.5%, -casein: 1.0%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: less than 200 = 3.0% Number of bright spots per cell: 10 or more = 5.0% Aggregated bright spot: none =

TABLE-US-00004 TABLE 4 Casein (-casein: 50%, -casein: 50%, -casein: 0%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: 200 to less than 300 = 3.0% Number of bright spots per cell: 10 or more = 5.0% Aggregated bright spot: none =

Extracted From Table

Experimental Example 3

[0143] The interstitial region was identified using Ventana I-VIEW PATHWAY HER2 (4B5) kit in the same manner as in (E1). Then, staining and evaluation were performed in the same manner as in (E2) to (E4), except that an anti-HER3 rabbit monoclonal antibody SP71 (manufactured by Abnova Corporation) was used in place of the anti-HER2 rabbit monoclonal antibody 4B5.

[0144] Consequently, results similar to those of Experimental Example 2 were obtained.

TABLE-US-00005 TABLE 5 Casein (natural) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: 300 to less than 500 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

TABLE-US-00006 TABLE 6 Casein (-casein: 45%, -casein: 45%, -casein: 10%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: less than 200 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

TABLE-US-00007 TABLE 7 Casein (-casein: 49%, -casein: 49%, -casein: 2%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: less than 200 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

TABLE-US-00008 TABLE 8 Casein (-casein: 50%, -casein: 50%, -casein: 0%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: 200 to less than 300 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

Experimental Example 4

[0145] A lung tissue array slide (tissue section-mounted glass slide; LC241b, manufactured by US Biomax, Inc.) was purchased, and the interstitial cell region was morphologically identified in the same manner as in (E1), except that an anti-PD-L1 rabbit monoclonal antibody SP142 (manufactured by Spring Bioscience (SBS) Corporation) was used. Then, staining and evaluation were performed in the same manner as in (E2) to (E4), except that an anti-PD-L1 rabbit monoclonal antibody SP142 (manufactured by Spring Bioscience (SBS) Corporation) was used in place of the anti-HER2 rabbit monoclonal antibody 4B5 and that the streptavidin-modified quantum dot-containing melamine resin nanoparticles prepared in Preparation Example 5 were used in place of the streptavidin-modified Texas Red dye-containing melamine resin particles. In (E), the wavelength of the irradiated excitation light was set at 415 to 455 nm using an excitation light optical filter (QD655-C, manufactured by OPTO-LINE, Inc.) installed in the fluorescence microscope, and the wavelength of the fluorescence to be observed was set at 648 to 663 nm using a fluorescence optical filter.

[0146] Consequently, results similar to those of Experimental Example 2 were obtained.

TABLE-US-00009 TABLE 9 Casein (natural) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: 300 to less than 500 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

TABLE-US-00010 TABLE 10 Casein (-casein: 47.5%, -casein: 47.5%, -casein: 5.0%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: less than 200 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

TABLE-US-00011 TABLE 11 Casein (-casein: 49%, -casein: 49%, -casein: 2%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: less than 200 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

TABLE-US-00012 TABLE 12 Casein (-casein: 50%, -casein: 50%, -casein: 0%) 1.0% 1.2% 2.4% 3.0% BSA 1.0% Interstitial noise: 200 to less than 300 = 3.0% Number of bright spots per cell: 5 or more 5.0% Aggregated bright spot: none =

Experimental Example 5

[0147] The steps of Experimental Example 1 were performed in the same manner, except that the process (E1) of identifying the interstitial cell region was excluded. That is, a breast cancer tissue array slide (tissue section-mounted glass slide; br243, manufactured by US Biomax, Inc.) was purchased and, after deparaffinization thereof, the tissue array slide was washed for replacement with water. The thus washed tissue array slide was subjected to a 15-minute autoclave treatment at 121 C. in 10 mM citrate buffer (pH 6.0), thereby performing an antigen retrieval treatment. After the retrieval treatment, the tissue array slide was washed with PBS and then subjected to a 1-hour blocking treatment with 1% BSA-containing PBS.

[0148] Immediately after performing (E2) in the same manner, hematoxylin staining was additionally performed for morphological observation, and (E3) and (E4) were subsequently performed in the same manner. As a result, it was confirmed that the number of bright spots per cell was 5 or more and that there was no aggregation of bright spots. The interstitial noise was not measured.

Preparation Example 6

Preparation of Biotin-Labeled BAC Probe

[0149] In accordance with the method described in Cell Biochem. Biophys. 2006; 45(1): 59, a nucleic acid molecule was prepared as described below. For 1 g (5 L) of a HER2-DNA clone (about 150 kbp) purchased from GSP Lab., Inc., dTTP of the HER2-DNA clone (nucleic acid molecule) was substituted with biotin-labeled dUTP by a nick translation method as described below in accordance with the protocol provided with a nick translation kit (product name: GSP-Nick Translation Kit K-015; manufactured by GSP Lab., Inc.).

[0150] Next, the resultant was allowed to react at 15 C. for 4 hours, and the reaction was terminated by heating at 70 C. for 10 minutes. Then, 25 L of distilled water was added to the centrifuge tube. The resulting reaction solution of a biotin-labeled BAC probe was purified using a micro-spin column for nucleic acid purification (MicroSpin S-200HR Column manufactured by GE Healthcare, product number: #27-5120-01). To this solution, about 5.56 L of 3 M sodium acetate solution (pH 5.2) and 150 L of 100% ethanol were added, and the resultant was left to stand at 20 C. for at least one hour and subsequently centrifuged at 4 C. and 16,000 rpm for 10 minutes to form precipitates. Further, 500 L of 70% ethanol was added, and the resultant was centrifuged at 4 C. and 16,000 rpm for 1 minute, followed by removal of the resulting supernatant. Thereafter, 5 to 10 L of distilled water was added to the thus formed precipitates and the precipitates were completely dissolved, thereby obtaining a solution of a biotin-labeled BAC probe having a final concentration of 1 g/250 L.

Experimental Example 6

[0151] The copy number of the HER2 gene was measured by FISH. As described below, FISH was carried out by performing deparaffinization, pretreatment of specimen slide, enzyme treatment, fixation of specimen, probe preparation, denaturation of DNA on specimen slide, hybridization, washing of glass slide and DAPI staining in the order mentioned.

Deparaffinization

[0152] A specimen slide of a HER2-positive staining control sample (HER2-FISH Control Slide manufactured by Pathology Institute Corp., code: PS-09006) was deparaffinized by sequentially performing the following treatments (1) to (4): (1) immersing the specimen slide in Hemo-De at normal temperature for 10 minutes; (2) immersing the specimen slide in fresh Hemo-De at normal temperature for 10 minutes, followed by three repetitions of the same operation; (3) immersing the specimen slide in 100% ethanol at room temperature for 5 minutes and washing the specimen slide twice, followed by dehydration; and (4) drying the specimen slide in the air or on a 45 to 50 C. slide warmer.

Pretreatment of Specimen Slide

[0153] In order to improve the reachability of the DNA probe, the specimen slide was pretreated by sequentially performing the following operations (1) to (6) to remove the proteins of cell membranes and nuclear membranes: (1) treating the specimen slide with 0.2 mol/L HCl at room temperature for 20 minutes; (2) immersing the specimen slide in purified water for 3 minutes; (3) immersing the specimen slide in a washing buffer (2 SSC: standard saline citrate) for 3 minutes; (4) immersing the specimen slide in a 80 C. pretreatment solution (1N NaSCN) for 30 minutes; (5) immersing the specimen slide in purified water for 1 minute; and (6) immersing the specimen slide in a washing buffer (2 SSC) for 5 minutes, followed by two repetitions of this immersion operation.

Enzyme Treatment

[0154] The thus pretreated specimen slide was subjected to an enzyme treatment by sequentially performing the following operations (1) to (4): (1) taking out the pretreated specimen slide and removing excess washing buffer by bringing the lower end of the glass slide into contact with a paper towel; (2) immersing the specimen slide in a protease solution heated to 37 C. for 10 to 60 minutes, which immersion process is desirably performed with 25 mg protease (in 50 mL of 2,500 to 3,000 units/mg of pepsin/1 M NaCl [pH 2.0] at 37 C. for 60 minutes) so as to degrade the proteins, particularly collagen, of cell membranes and nuclear membranes; (3) immersing the specimen slide in a washing buffer for 5 minutes, followed by two repetitions of this operation; and (4) drying the specimen slide in the air or on a 45 to 50 C. slide warmer for 2 to 5 minutes.

Fixation of Specimen

[0155] For fixation of the specimen, the pretreated specimen slide was subjected to the following treatments (1) to (3): (1) immersing the specimen slide in 10% neutral buffered formalin (4% paraformaldehyde-phosphate buffer manufactured by Wako Pure Chemical Industries, Ltd., product number: 163-20145) at normal temperature for 10 minutes; (2) immersing the specimen slide in a washing buffer for 5 minutes, followed by two repetitions of the same operation; and (3) drying the specimen slide in the air or on a 45 to 50 C. slide warmer for 2 to 5 minutes.

Probe Preparation

[0156] A solution of the DNA probe prepared in Preparation Example 6, which had been freeze-stored, was thawed back to room temperature, and the viscosity of the solution was sufficiently reduced to such a level at which an exact volume of the solution can be collected by pipette operation, after which the solution was mixed using a vortex mixer or the like.

Denaturation of DNA on Specimen Slide

[0157] For denaturation of DNA on the specimen slide, the thus specimen-fixed specimen slide was subjected to the following treatments (1) to (8): (1) prior to the preparation of the specimen slide, placing and preheating a moist box having a water-moistened paper towel on the bottom (a hermetic container whose side surfaces are taped with paper towel) in a 37 C. incubator; (2) confirming that a denaturation solution (70% formamide/SSC [150 mM NaCl, 15 mM sodium citrate]) has a pH of 7.0 to 8.0 at normal temperature, placing the denaturation solution in a Coplin jar and heating the Coplin jar in a warm water bath until the solution temperature reaches 72 C.1 C. (leaving the Coplin jar in a 721 C. warm water bath for at least 30 minutes); (3) marking a region on the back side of the specimen with a circle using a diamond pen or the like to clearly indicate a hybridization region; (4) immersing the specimen slide in the 721 C. denaturation solution placed in the Coplin jar to denature the DNA on the specimen slide; (5) taking out the specimen slide from the denaturation solution using a forceps, immediately placing the specimen slide in 70% ethanol at room temperature, shaking the slide for removal of formamide and leaving the specimen slide immersed for 1 minute; (6) taking out the specimen slide from the 70% ethanol, placing the specimen slide in 85% ethanol, shaking the slide for removal of formamide and leaving the specimen immersed for 1 minute, followed by two repetitions of the same operations using 100% ethanol; (7) removing ethanol by bringing the lower end of the specimen glass slide into contact with a paper towel, and then wiping the back side of the glass slide with a paper towel; and (8) drying the specimen slide using a dryer or on a 45 to 50 C. slide warmer for 2 to 5 minutes.

Hybridization

[0158] The thus denaturation-treated specimen slide was subjected to hybridization with 10 L (10 to 50 ng) of the above-prepared DNA probe by sequentially performing the following treatments (1) to (3): (1) adding 10 L of the above-prepared DNA probe to the hybridization region of the specimen slide and immediately placing a 22 mm22 mm cover glass over the probe to uniformly spread the probe while preventing air bubbles from entering the hybridization region; (2) sealing the cover glass with paper bond; and (3) placing the specimen slide in the previously heated moist box, placing the lid and then performing hybridization in a 37 C. incubator for 14 to 18 hours.

Washing of Glass Slide

[0159] The thus hybridized specimen slide was washed by sequentially performing the following treatments (1) to (6): (1) placing a post-hybridization washing buffer (2 SSC/0.3% NP-40) in a Coplin jar and preheating the Coplin jar in a warm water bath until the temperature of the post-hybridization washing buffer reaches 72 C.1 C. (leaving the Coplin jar in a 721 C. warm water bath for at least 30 minutes); (2) preparing another Coplin jar containing the post-hybridization washing buffer and maintaining it at normal temperature; (3) removing the paper bond seal using a forceps; (4) immersing the specimen slide in this post-hybridization washing buffer until the cover glass spontaneously comes off in the solution; (5) taking out the specimen slide from the solution, removing excess solution and then immersing the specimen slide in the post-hybridization washing buffer heated to 721 C. for 2 minutes, which immersion treatment is desirably performed at a temperature of 73 C. or lower for a period of 2 minutes or less; and (6) taking out the specimen slide from the Coplin jar and air-drying the specimen slide in shade (for example, in a closed drawer or on a shelf of a closed cabinet).

Fluorescent Labeling Treatment of Probe

[0160] To the biotin-labeled DNA probe bound with HER2 gene, the streptavidin-bound fluorescent substance-containing melamine nanoparticles of Preparation Example 3 were bound as follows.

[0161] The particles prepared in Preparation Example 3 were diluted with a diluent to a concentration of 0.02 nM, and 100 L of the resultant was dropped onto the specimen slide to allow a binding reaction to take place at room temperature for 60 minutes. The specimen slide was subsequently washed three times by immersion in PBS for 5 minutes. A case where 1% BSA was used as the diluent was compared with a case where a mixture of caseins (composition, -casein (c6780, manufactured by Sigma-Aldrich): 50%, -casein (c6905, manufactured by Sigma-Aldrich): 50%) and BSA was used as the diluent.

DAPI Staining

[0162] DAPI staining was performed as follows. First, 10 L of a DAPI counter-staining liquid was added to the hybridization region of the specimen slide. Next, after subjecting the specimen slide to hybridization, in order to count the number of cells, cell nuclei were stained by performing DAPI staining (2 g/mL PBS) at 25 C. for 10 minutes, and a cover glass was placed on the specimen slide. This specimen slide was stored in shade until signal measurement. As DAPI (4,6-diamidino-2-phenylindole dihydrochloride), D1306 manufactured by Molecular Probes Inc. was used.

Observation

[0163] The specimen slide subjected to FISH as described above was observed in the following manner.

Observation Under Fluorescence Microscope

[0164] As for observation under a fluorescence microscope, the section subjected to FISH as described above was observed (at 600 magnification) under a fluorescence microscope Zeiss Imager (camera: MRm monochrome camera with cooling function, objective lens: 60 oil immersion lens) at a magnification of 600 to obtain fluorescence images (static fluorescence images) and to measure the number of bright spots.

[0165] As a result, comparing to the case where 1% BSA was used as the particle diluent, the number of bright spots (the number of bright spots per cell in the HER2 3+ region (that is, cancer cell region)) was found to be doubled in those cases where a mixed liquid containing 2.4% of a casein mixture (composition=-casein (c6780, manufactured by Sigma-Aldrich): 50%, -casein (c6905, manufactured by Sigma-Aldrich): 50%) and 1 to 5% of BSA was used.