JP6658330B2 - How to prevent dissociation of fluorescent nanoparticles from tissue sections - Google Patents

Description

  The present invention relates to a method for preventing the dissociation of fluorescent nanoparticles bound to a target biological substance in a tissue section prepared as a stained slide used for pathological diagnosis or the like. More specifically, the present invention relates to a process for fixing a slide stained by an immunostaining method using fluorescent nanoparticles or a fluorescent in situ hybridization (FISH) method using an immobilizing reagent for fluorescent nanoparticles.

  In pathological diagnosis, slides are prepared by slicing a specimen collected from a patient, and based on the stained image when stained by a predetermined method, the morphology of cells or tissues is observed, and the expression level of a specific biological substance is determined. Is a method for diagnosing various events such as whether or not the patient is suffering from a specific disease or whether or not a specific therapeutic drug is effective by quantifying and evaluating the above.

  For example, using a specimen prepared by collecting a cancer tissue, a HER2 gene (HER2 / neu, c-erbB-2), which is a kind of an oncogene, and / or a membrane protein produced from the HER2 gene, The prognosis of breast cancer patients can be diagnosed by quantifying and evaluating HER2 protein, which is presumed to function as a cell growth factor receptor, and the molecular target therapeutic agent trastuzumab (trade name “Herceptin” (registered) (Trademark), anti-HER2 monoclonal antibody), and the like, and pathological diagnosis for predicting the therapeutic effect is widely performed. In human breast cancer cases, HER2 gene amplification and HER2 protein overexpression are observed in 15 to 25%, but HER2 overexpression in cancer cells basically occurs with gene amplification at the DNA level. HER2 test methods for cancer tissues are classified into a method of observing amplification at the DNA level, a method of observing overexpression at the RNA level, and a method of observing overexpression at the protein level. Representative methods for testing at the protein level and at the DNA level are immunostaining or immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Such HER2 test is regarded as clinically important, and the standard procedure and criteria (score) of HER2 test by immunostaining (IHC method) and FISH method are defined by the 2007 ASCO / CAP guidelines. ing.

  The FISH method is a method for detecting the copy number of the HER2 gene in the metaphase nucleus of a cancer cell using a fluorescently labeled DNA probe for the HER2 gene on a tissue section embedded in formalin-fixed paraffin. Generally, a slide (specimen) on which a sliced formalin-fixed, paraffin-embedded tissue section is placed is prepared, deparaffinized, etc., so as to be suitable for hybridization, and then reacted with a fluorescent-labeled probe. To the HER2 gene in the nucleus (on the chromosome). Usually, the nucleus (chromosome) is further stained with a fluorescent dye DAPI (4 ', 6-diamidino-2-phenylindole) which intercalates into double-stranded DNA. The tissue section stained in this way is subjected to encapsulation using an encapsulant containing an anti-fading agent, and then a fluorescence image is taken while irradiating a predetermined excitation light. A stained image is taken. The photographed HER2 gene-labeled fluorescence image and the DAPI-stained image are superimposed, and the number of bright spots observed in the nucleus (on the chromosome) per cell is measured. Is amplified or not.

  In the FISH method as described above, for the purpose of fluorescent labeling of a target gene (nucleic acid), a fluorescent substance such as a high-brightness fluorescent substance such as a quantum dot or a fluorescent substance such as a quantum dot or a fluorescent dye is used. It is preferable to use fluorescent dye-integrated nanoparticles in which the body is integrated with a resin or the like. For such an embodiment, for example, reference can be made to Patent Document 1 (International Patent Publication WO2015 / 141856).

  On the other hand, the immunostaining method (IHC method) is a method for detecting the amount of HER2 protein expressed in a cell membrane on a formalin-fixed paraffin-embedded tissue section using a fluorescently labeled anti-HER2 antibody. The conventional immunostaining method (original IHC method) employs a method using an anti-HER2 antibody labeled with an enzyme that generates a dye when a predetermined substrate is added (enzyme antibody method). An immunostaining method (fluorescent antibody method) using an anti-HER2 antibody labeled with a fluorescent substance having excellent properties has also been used. As in the case of the FISH method, a slide (specimen) on which a sliced formalin-fixed paraffin-embedded tissue section is mounted is prepared, deparaffinized, and the like, and is made suitable for immunostaining. Then, a fluorescently labeled antibody is reacted to bind to the HER2 protein on the cell membrane.

  In the case of using the fluorescent nanoparticles as described above for fluorescent labeling, unlike the IHC method using a conventional enzyme, the dye does not mix, so that the immunostaining method can be used on a single tissue section. In addition to the treatment, a staining treatment for observing cell morphology can be performed simultaneously. For example, when hematoxylin / eosin staining (HE staining), which is standardly used for observing the morphology of cells or tissues, is used, the cell nucleus / calcium / cartilage tissue / bacteria / mucus is blue-blue to pale by hematoxylin staining. It is stained blue, and cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are stained red to dark red by eosin staining. The tissue section immunostained in this manner is subjected to encapsulation using an encapsulant containing an anti-fading agent, as in the case of the FISH method, and a fluorescence image is taken while irradiating a predetermined excitation light, Further, a stained image for morphological observation is taken in a bright field (note that eosin emits fluorescence, so a stained image of eosin can be taken as a fluorescence image). Then, the photographed fluorescence image labeled with the HER2 protein and a (fluorescence) image for observing the morphology of the cell, which can identify the position of the cell membrane, are superimposed to obtain a bright spot observed in the cell membrane region per cell. The number is measured, and it is determined from the value whether or not the HER2 protein is abnormally expressed.

  Patent Document 2 (International Patent Publication WO2013 / 035688) discloses that, as described above, HE staining is performed on the same tissue section, and a target biological substance is fluorescently labeled with a fluorescent substance such as fluorescent nanoparticles. A tissue staining method is described. Patent Document 3 discloses a tissue staining method in which HE staining is performed on the same tissue section, and different target biological substances are fluorescently labeled using two or more kinds of fluorescent nanoparticles having different average particle sizes. Has been described.

International Publication WO2015 / 141856 pamphlet International Publication WO2013 / 035688 Pamphlet JP-A-2005-117980

  The fluorescent nanoparticles used in the conventional immunostaining method or the tissue evaluation method by the FISH method are used in combination with a staining solution (for example, an HE solution which is a staining solution for morphological observation: a solution of hematoxylin and / or eosin, a DAPI solution). In this case, there is a problem that a part of the fluorescent nanoparticles specifically bound to the target biological substance is dissociated, and the number of fluorescent luminescent spots of the fluorescent nanoparticles decreases in observation of a microscope image. In particular, in the case of performing HE (both hematoxylin and eosin) staining, the number of particles is reduced by about 20 to 50% as compared with the case of performing only H (hematoxylin) staining, that is, E (eosin). A phenomenon in which some of the fluorescent nanoparticles are dissociated remarkably by staining is observed. Dissociation of the fluorescent nanoparticles occurs to a greater or lesser extent in cases other than E staining such as nuclear staining with a DAPI solution. It became clear that there was.

  Patent Document 2 cited above discloses that, when performing an HE staining step after an immunostaining step using fluorescent nanoparticles, from the viewpoint of suppressing a decrease in fluorescence intensity from a fluorescent substance introduced into a tissue section as a label, It is described that it is preferable to perform a fixing treatment after the immunostaining step and before the HE staining step (see paragraph [0085]). However, the fixing treatment described in Patent Literature 2 is such that the fluorescence emitted from the phosphor is relatively weakened due to the effect of autofluorescence derived from HE staining (especially eosin), thereby preventing the identification of the phosphor. This is a means taken together with the adjustment of the wavelength at the time of observation (see paragraph [0097]) in order to avoid. In addition, in a pathological region, it is generally known to use an immobilization reagent for the purpose of preventing cells from detaching from a slide, and formalin or the like generally used in Patent Document 2 is also used. (See paragraph [0086]). Patent Literature 2 does not disclose a process for preventing a fluorescent substance such as fluorescent nanoparticles from dissociating from a target biological substance and reducing the number of fluorescent luminescent spots.

  In the invention described in Patent Document 3, tissue sections are immunostained with the first fluorescent particles, and a blocking treatment is performed using a PBS buffer containing BSA or the like, and then the second fluorescent Immunostaining of tissue sections with particles is performed. When the immunostaining is performed a plurality of times on a single sample slide (tissue section) as described above, it is preferable that a blocking process is performed between the staining processes to suppress nonspecific adsorption. However, even when the blocking treatment is performed, a part of the fluorescent nanoparticles specifically bound to the target biological substance is dissociated, and there is a problem that the number of fluorescent spots of the fluorescent nanoparticles decreases in the observation of the microscope image. Was.

  Further, even when the treatment for staining with a morphological observation staining solution or the blocking treatment is not performed, the washing treatment using a PBS solution or the like is usually performed after performing the fluorescent labeling treatment. A part of the fluorescent nanoparticles specifically bound to the substance is dissociated, and there is a concern that the number of fluorescent luminescent spots of the fluorescent nanoparticles decreases in the observation of a microscope image. Such a dissociation phenomenon may be caused by immunostaining with a fluorescent dye or FISH method. However, in the case of staining with fluorescent nanoparticles, the bulkiness of the particles and the surface condition of the particles have an effect. It is presumed that it appears as a remarkable phenomenon.

  The present invention provides a process of labeling a target biological substance with fluorescent nanoparticles by immunostaining or FISH on a single sample slide (tissue section), followed by a process using various solutions, for example, When performing staining with a staining solution for cell morphology observation according to the staining method of the target biological material, blocking treatment, washing treatment, etc., the fluorescent nanoparticles labeled with the target biological material are dissociated and observed under a microscope It is an object to provide a method capable of preventing the number of fluorescent luminescent spots from being reduced.

  The present inventors have conducted intensive studies to solve the problems of the present invention, and as a result, fixed treatment using a fluorescent nanoparticle fixing reagent on a fluorescently labeled tissue section (fluorescent nanoparticle fixing treatment) By performing the above, the fluorescent nanoparticle labeled with the target biological substance can be obtained even if a staining treatment using a morphological observation staining solution such as an HE solution or a DAPI solution is performed, or a blocking treatment or a washing treatment is performed. It has been found that dissociation can be suppressed. This is because fluorescent nanoparticles are dissociated by immobilizing reagents used in the present invention by cross-linking fluorescent nanoparticles with tissue sections (proteins around the target biological material, etc.) or cross-linking proteins on tissue sections. Even so, it is presumed that the fluorescent nanoparticles are fixed by staying around the target biological material.

That is, the present invention includes the invention represented by the following embodiments.
[Item 1]
Processing for labeling target biological substances contained in tissue sections on specimen slides with fluorescent nanoparticles by immunostaining or FISH (fluorescent labeling)
And fixing the tissue section subjected to the fluorescent labeling treatment with a fluorescent nanoparticle fixing reagent (fluorescent nanoparticle fixing treatment)
A method for preventing dissociation of fluorescent nanoparticles from a tissue section.
[Item 2]
Item 4. The method according to Item 1, further comprising a step of staining (staining) and / or blocking the tissue section on which the fluorescent nanoparticle fixing treatment has been performed, with a staining solution for cell morphology observation.
[Item 3]
Item 3. The method according to Item 1 or 2, wherein the immobilizing reagent is selected from compounds having an NHS group (N-hydroxysuccinimide group) and / or an epoxy group as a functional group.
[Item 4]
Item 3. The method according to Item 1 or 2, wherein the immobilization reagent is selected from NHS-PEG-NHS, biotin-PEG-NHS, and epoxy-PEG-epoxy.
[Item 5]
Item 5. The method according to any one of Items 1 to 4, wherein the concentration of the immobilization reagent is 1 μM to 500 μM.
[Item 6]
Item 6. The method according to any one of Items 1 to 5, wherein the staining solution is selected from hematoxylin, eosin and DAPI.
[Item 7]
Item 7. The method according to any one of Items 1 to 6, wherein the fluorescent nanoparticles are fluorescent dye-integrated nanoparticles or inorganic fluorescent nanoparticle aggregates, and have an average particle diameter of 40 nm to 160 nm.

  According to the method of the present invention, the stained slide that has been subjected to the fluorescent labeling treatment may be stained for morphological observation using a staining solution (hematoxylin-eosin, DAPI, etc.), or may be subjected to blocking treatment, washing treatment, or the like. Dissociation of the fluorescent nanoparticles from the slide can be suppressed. Therefore, the number of bright spots of the fluorescent nanoparticles during microscopic observation does not decrease, and the accuracy of quantification of the target biological substance can be improved.

FIG. 1 is a flowchart illustrating an example of an embodiment of a method for preventing dissociation of fluorescent nanoparticles from a tissue section according to the present invention in the first embodiment when performing staining based on an immunostaining method. FIG. 2 is a flowchart showing an example of a second embodiment of the method for preventing dissociation of fluorescent nanoparticles from a tissue section according to the present invention in the case of performing staining based on the FISH method. FIG. 3 shows [A] a fluorescent image taken in Example 1 and [B] a fluorescent image taken in Comparative Example 1. FIG. 4 shows [A] a fluorescent image taken in Example 3 and [B] a fluorescent image taken in Comparative Example 3. FIG. 5 is [A] a hematoxylin-eosin stained image taken in Example 3, and [B] a hematoxylin-eosin stained image taken in Comparative Example 3.

  The method for preventing the dissociation of the fluorescent nanoparticles from the tissue section prepared as the stained slide of the present invention is an embodiment performed based on an immunostaining method (hereinafter, referred to as “first embodiment”), and FISH. An embodiment performed based on the law (hereinafter, referred to as a “second embodiment”) can be roughly classified.

  In both the first embodiment and the second embodiment, the entire process for producing a stained slide can be mainly classified into a “sample preparation process”, a “staining process”, and a “sample preparation process”.

  The sample pretreatment step of the first embodiment of the present invention relating to the immunostaining method generally includes a deparaffinization treatment, an antigen activation treatment, a washing treatment, and, if necessary, a cell fixing treatment.

  In the staining step of the first embodiment, a process of performing fluorescent labeling based on an immunostaining method (immunostaining process), that is, a primary process depending on whether the target biological substance is directly labeled or indirectly labeled. Antibody treatment, secondary antibody treatment, fluorescent nanoparticle fixing treatment, morphological observation staining treatment, and, if necessary, washing treatment and blocking treatment performed before and after each treatment are included.

  The sample post-processing process of the first embodiment includes a sealing process, and if necessary, a clearing process and a dehydrating process.

  The sample pretreatment step of the second embodiment of the present invention relating to the FISH method generally includes a deparaffinization treatment, a pretreatment for FISH, an enzyme treatment, and if necessary, a cell fixation treatment.

  The staining process of the second embodiment includes a process of performing fluorescent labeling based on the FISH method (FISH process), that is, a DNA denaturation process, a hybridization process, a post-hybridization washing process, and a process for fixing fluorescent nanoparticles and observing morphology. A staining treatment (for example, nuclear staining with DAPI), and a washing treatment and a blocking treatment performed before and after each treatment as necessary are included.

  The sample post-processing process of the second embodiment includes a sealing process, a solvent replacement process and a dehydration process as necessary.

  Hereinafter, the processing required to implement the present invention will be further described. Items not specifically described herein, for example, general items of steps and processes required to complete slides stained based on the FISH method or immunostaining method, and completed stained slides The observation / photographing process used, the image processing / analysis process using the photographed image, and the like are appropriate according to the descriptions in Patent Documents 1 to 3 and other general or known technical matters. It can be.

(Fluorescent nanoparticles)
In the present invention, fluorescent nanoparticles are used as a substance for fluorescently labeling a target gene or protein. The fluorescent nanoparticle is a nanoparticle (1 μm or less in diameter) particulate phosphor, and can emit fluorescence having sufficient luminance with one particle. What is necessary is just to select fluorescent nanoparticles that emit fluorescence (color) of a desired wavelength in a captured fluorescent image. Further, when there are a plurality of biological target substances to be subjected to fluorescent labeling, a plurality of types of fluorescent nanoparticles that emit fluorescence of different wavelengths corresponding to the respective substances may be used in combination. Such fluorescent nanoparticles can be broadly classified into inorganic fluorescent nanoparticles, inorganic fluorescent nanoparticle aggregates, and fluorescent dye integrated nanoparticles.

(Inorganic fluorescent nanoparticles)
In the present invention, quantum dots or silica dots can be used as the inorganic fluorescent nanoparticles. These inorganic fluorescent nanoparticles can constitute the brightness of a luminescent spot having an observable luminance alone without preparing an inorganic fluorescent nanoparticle assembly as described below.

  As the quantum dot, a quantum dot containing a II-VI group compound, a III-V compound, or a group IV element as a component ("II-VI quantum dot", "III-V quantum dot", " Group IV quantum dot "). Specifically, a particle dot such as CdSe exemplified in International Publication WO2012 / 133047 can be mentioned. Any of these inorganic fluorescent nanoparticles may be used alone or in combination of two or more.

  Alternatively, a quantum dot having the above-described quantum dot as a core and a shell provided thereon may be used. Hereinafter, as the notation of the quantum dot having the shell, when the core is CdSe and the shell is ZnS, it is described as CdSe / ZnS. Specific examples include, but are not limited to, CdSe / ZnS exemplified in International Publication WO2012 / 133047.

  Quantum dots may be surface-treated with an organic polymer or the like, if necessary. For example, commercially available CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen) and CdSe / ZnS having a surface amino group (manufactured by Invitrogen) can be used.

(Inorganic fluorescent nanoparticle assembly)
Inorganic fluorescent nanoparticle aggregates include multiple inorganic fluorescent nanoparticles, such as quantum dots or silica dots, encapsulated in a parent substance or attached to the surface, or linked to individual inorganic fluorescent nanoparticles. This is a nano-sized (1 μm or less in diameter) particulate phosphor that has been integrated. The use of such inorganic fluorescent nanoparticle aggregates in immunostaining is more efficient than the use of inorganic fluorescent nanoparticles alone (one particle without integration), in which the fluorescence of the target biomolecule is labeled. It is preferable because the intensity of the fluorescence emitted per label can be enhanced, noise such as autofluorescence of cells and the like and discrimination from other dyes can be enhanced, and discoloration due to irradiation with excitation light can be suppressed. .

  Examples of the inorganic fluorescent nanoparticles to be integrated in the inorganic fluorescent nanoparticle assembly include the same inorganic fluorescent nanoparticles that can be used alone as described above.

  As an example of the embodiment of the inorganic fluorescent nanoparticle aggregate, an inorganic fluorescent nanoparticle integrated resin particle manufactured by using an inorganic fluorescent nanoparticle as a fluorescent material and a resin as a matrix; using an inorganic fluorescent nanoparticle as a fluorescent material And inorganic fluorescent nanoparticle-integrated silica particles produced using silica as a matrix.

  As a base constituting the inorganic fluorescent nanoparticle aggregate, a substance such as a resin or silica capable of integrating a fluorescent substance with physical or chemical bonding force can be used. Examples of the resin include thermosetting resins such as melamine resin, urea resin, benzoguanamine resin, phenol resin, and xylene resin; and styrene resin, (meth) acrylic resin, polyacrylonitrile, and AS resin (acrylonitrile-styrene copolymer). And various kinds of homopolymers and copolymers produced using one or more kinds of monomers such as ASA resin (acrylonitrile-styrene-methyl acrylate copolymer).

  Furthermore, as described later in the section “Average particle diameter of inorganic fluorescent nanoparticle aggregates and fluorescent dye-containing nanoparticles” (size selective precipitation method), an aggregate of quantum dots using an adsorbate having a lipophilic group is used. Or an aggregate of silica dots can be obtained by stopping the end cap in an incomplete state.

(Fluorescent dye integrated nanoparticles)
Fluorescent dye-integrated nanoparticles are nanosized (1 μm or less in diameter) particle-like phosphors that are integrated by enclosing a plurality of fluorescent dyes in a parent substance or attaching them to the surface. . The use of such a fluorescent dye-incorporated nanoparticle in immunostaining is different from the case where a fluorescent dye is used alone (in a single molecule without integration) in that a fluorescent label 1 labeled with a target biomolecule is used. This is preferable because the intensity of the fluorescence emitted from each end can be enhanced, the distinction from noise such as autofluorescence of the cells and other dyes can be enhanced, and the fading due to irradiation with excitation light can be suppressed.

  Examples of the fluorescent dye to be integrated in the fluorescent dye-containing nanoparticles include, for example, fluorescein dye, rhodamine dye, Alexa Fluor (registered trademark, manufactured by Invitrogen) -based dye, BODIPY (registered trademark, manufactured by Invitrogen) -based dye, cascade (Registered trademark, Invitrogen Corporation) dyes, coumarin dyes, NBD (registered trademark) dyes, pyrene dyes, cyanine dyes, perylene dyes, oxazine dyes, and other low-molecular-weight organic compounds (polymers such as polymers) A non-compound). Above all, rhodamine-based dyes such as sulforhodamine 101 and its hydrochloride, TexasRed (registered trademark), and perylene-based dyes such as perylenediimide are preferable because of their relatively high light resistance.

  Examples of the embodiment of the fluorescent dye-integrated nanoparticles include a fluorescent dye-integrated resin particle manufactured using a fluorescent dye as a fluorescent substance and a resin as a base; using a fluorescent dye as a fluorescent substance, and using silica as a base. Fluorescent dye-integrated silica particles to be produced are exemplified.

  For example, fluorescent dye-integrated resin particles produced using a fluorescent dye such as perylenediimide, sulforhodamine 101 or its hydrochloride (Texas Red) as a fluorescent material, and using a resin such as a melamine resin or a styrene resin as a base material are labeled. Because of their excellent performance and the like, they are preferred as the fluorescent dye-integrated nanoparticles in the present invention.

Examples of the matrix constituting the fluorescent dye-integrated nanoparticles include the same as the inorganic fluorescent nanoparticle aggregates described above.
In particular, a melamine resin or a styrene resin is preferable because nanoparticles in which a fluorescent substance such as a fluorescent dye is integrated can be easily produced and nanoparticles having high emission intensity can be obtained.

  Inorganic fluorescent nanoparticle aggregates and fluorescent dye-containing nanoparticles as described above are known, and details of phosphors used for the production thereof, a matrix, a production method, and specific examples of embodiments are described in, for example, International Publication WO2013 / 2013. See, for example, pamphlet No. 035703, pamphlet of International Publication WO2013 / 147081, pamphlet of International Publication WO2014 / 136776, and the like.

(Average particle diameter of inorganic fluorescent nanoparticle aggregates and fluorescent dye integrated nanoparticles)
In the present invention, when an inorganic fluorescent nanoparticle aggregate or a fluorescent dye-integrated nanoparticle is used as the fluorescent nanoparticle, as the particle size of the fluorescent nanoparticle is reduced, the specific surface area is increased, and the bonding force with the tissue section is increased. Therefore, the average particle diameter of the inorganic fluorescent nanoparticle aggregate or the fluorescent dye-integrated nanoparticle is preferably 40 nm or more and 160 nm or less.

  The particle diameter of the inorganic fluorescent nanoparticle aggregate or the fluorescent dye-integrated nanoparticle corresponds to a measured value obtained by taking an image using a scanning electron microscope (SEM) and measuring the cross-sectional area of the fluorescent labeling resin particle. It can be measured as a diameter as a circle area (diameter equivalent to an area circle). The average particle size and the coefficient of variation for the population of inorganic fluorescent nanoparticle aggregates or fluorescent dye-loaded nanoparticles are determined above for each of a sufficient number (eg, 1000) of inorganic fluorescent nanoparticle aggregates or fluorescent dye-loaded nanoparticles. After measuring the particle diameter as described above, the average particle diameter is calculated as its arithmetic average, and the coefficient of variation is calculated by the formula: 100 × standard deviation of particle diameter / average particle diameter.

  The average particle diameter of the inorganic fluorescent nanoparticle aggregate or the fluorescent dye integrated nanoparticle can be adjusted to a desired range by adjusting the conditions in the production method.

  As an example of a method for producing the fluorescent dye-integrated nanoparticles, an emulsion polymerization method, that is, a (co) monomer for synthesizing a resin (a thermoplastic resin or a thermosetting resin) forming a base of the fluorescent dye-integrated nanoparticles is (co) A method of adding a phosphor while polymerizing and incorporating the phosphor into the inside or the surface of the (co) polymer is exemplified. In the reaction system in the emulsion polymerization method, the surfactant forms a micelle having an aqueous phase on the outside and an oil phase on the inside, and the oil phase inside the micelle contains a monomer constituting the resin, and the micelle is formed. The polymerization reaction is performed inside.

  When synthesizing the fluorescent dye-integrated nanoparticles by such an emulsion polymerization method, for example, by adding a surfactant having an emulsifying action in an amount of 10 to 60% by weight based on the resin raw material, the average particle diameter is 30 nm to 300 nm particles can be produced. When the proportion of the surfactant is increased, smaller particles can be produced, and the average particle diameter can be reduced to 30 nm or less. Conversely, if the proportion of the surfactant is reduced, larger particles can be produced, and the average particle diameter can be 300 nm or more. In addition, when the amount of the surfactant used is constant, the ratio of the fluorescent dye-incorporated nanoparticles to the total reaction system of the resin raw material and the phosphor used in the production of the fluorescent dye-incorporated nanoparticles is also changed. The average particle size can be adjusted.

  On the other hand, regarding the adjustment of the average particle diameter of the inorganic fluorescent nanoparticle aggregate, after the inorganic fluorescent nanoparticle aggregate is manufactured, the inorganic fluorescent nanoparticle aggregate is classified by a size selective precipitation method, and the inorganic fluorescent nanoparticle aggregate having a predetermined average particle size is recovered. It can be done by doing.

  The size-selective precipitation method is to first adsorb an adsorbate having a lipophilic group on the surface of inorganic fluorescent nanoparticles such as quantum dots, then disperse the inorganic fluorescent nanoparticles in a lipophilic solvent, Is added little by little to cause aggregation and accumulation. Since the dispersibility of inorganic fluorescent nanoparticles strongly depends on the interaction between the adsorptive groups on the surface of the nanoparticles and the solvent, by gradually adding the additive, aggregated precipitates are formed in order from large-sized inorganic fluorescent nanoparticles. This is because the precipitate is collected by centrifugation and redispersed in a solvent to form a monodisperse inorganic fluorescent nanoparticle aggregate having a narrow distribution.

  Examples of the adsorbate having a lipophilic group include compounds having an alkyl group such as heptane, octane and dodecane, and those having 8 to 12 carbon atoms are preferable.

  Examples of the lipophilic solvent include pyridine and hexane, and examples of the amphiphilic additive include chloroform and methanol.

  Examples of groups that can be adsorbed on the surface of inorganic fluorescent nanoparticles such as quantum dots include phosphino groups such as trioctylphosphine (TOP), phosphine oxide groups such as trioctylphosphine oxide (TOPOT), phosphate groups, and amino groups. Are known and can be subjected to a size-selective precipitation method to form inorganic fluorescent nanoparticles coated with an adsorbate having a lipophilic group.

  On the other hand, silica dots are inorganic fluorescent nanoparticles having a particle size of about 7 to 11 nm, which are produced by a Stover method or the like in which condensation is performed using tetraethoxysilane (TEOS). The HO-Si group present on the particle surface can be end-capped with alkyltrimethoxysilane. At this time, if the end cap is stopped in an incomplete state, the particles will be bonded to the Si-O-Si polymer. You can obtain a chained aggregate. The removal of the solvent and the adsorbate from the surface of the collected inorganic fluorescent nanoparticle assembly can be performed by a known heat treatment.

<Fluorescent labeling (immunostaining)>
The fluorescent labeling process performed in the first embodiment of the present invention is a process of labeling a target protein with fluorescent nanoparticles based on an immunostaining method in a staining step.

  When performing the labeling treatment based on the immunostaining method, the specimen slide that has been subjected to the activation treatment is immersed in a labeling solution for immunostaining, and one or more labeling agents in the labeling solution are directly or indirectly targeted. It is bound to a protein (antigen) and labeled.

  The target protein (antigen) is not particularly limited, but typically, a gene that can be a target of pathological diagnosis based on an immunostaining method, for example, HER2, TOP2A, HER3, EGFR, P53, MET, ki67, other genes From proteins derived from various cancer / tumor-related genes (so-called biomarker genes), as well as cancer-related proteins such as cancer growth factors, transcription factors, growth factor receptors, and transcription factor receptors. You can choose. Therefore, the antibody described below can be prepared based on a known technique as having the binding ability suitable for the target protein (antigen) as described above, and can also be obtained as a commercial product.

  Any antibody as a substance that specifically binds to the target biological substance (and a reference biological substance to be described later) may be a polyclonal antibody, but a monoclonal antibody is preferred from the viewpoint of quantitative stability. The type of animal that produces the antibody (immunized animal) is not particularly limited, and may be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like, as in the related art.

The antibody is not a natural full-length antibody but may be an antibody fragment or derivative as long as it has the ability to specifically recognize and bind to a specific biological substance (antigen). That is, the term “antibody” as used herein includes not only full-length antibodies, but also antibody fragments such as Fab, F (ab) ′ ₂ , Fv, scFv, chimeric antibodies (humanized antibodies, etc.), multifunctional antibodies And the like.

There are various methods for immunostaining, and it is not particularly limited as long as a tissue section can be stained so that a target protein can be fluorescently labeled and used for pathological diagnosis or the like. These include:
A method of preparing a fluorescent-labeled primary antibody in which fluorescent nanoparticles and a primary antibody are linked, and directly labeling and staining a target protein with the fluorescent-labeled primary antibody (primary antibody method);
Preparing a primary antibody and a fluorescent-labeled secondary antibody obtained by linking a fluorescent nanoparticle and a secondary antibody, reacting the primary antibody with the target protein, and then reacting the primary antibody with the fluorescent-labeled secondary antibody Method for indirectly fluorescently labeling and staining the target protein (secondary antibody method)
A primary antibody and biotin-modified primary antibody linked to biotin, and a fluorescent nanoparticle and avidin-modified fluorescent nanoparticle linked to avidin or streptavidin are prepared. After the biotin-modified primary antibody is reacted with the target protein, A method of reacting avidin-modified fluorescent nanoparticles and indirectly fluorescently labeling and staining a target protein using an avidin-biotin reaction (avidin-biotin combined primary antibody method);
A primary antibody, a biotin-modified secondary antibody in which biotin is linked to biotin, and an avidin-modified fluorescent nanoparticle in which fluorescent nanoparticles are linked to avidin or streptavidin are prepared, and the primary antibody is reacted with the target protein. A method of reacting a biotin-modified secondary antibody, followed by further reacting with avidin-modified fluorescent nanoparticles, and indirectly fluorescently labeling and staining the target protein using an avidin-biotin reaction (avidin-biotin secondary antibody) Law).

  In the above-described avidin-biotin combined primary antibody method or avidin-biotin combined secondary antibody method, biotin and avidin may be replaced by a hapten (having a relatively low molecular weight that has no immunogenicity but is antigenic and can react with the antibody). Combinations of anti-hapten antibodies such as dicoxigenin and anti-dicoxygenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antigens, as well as other substances having similar specific reactivity.

  The immunostaining method may be performed according to standard procedures and processing conditions for each of the various methods described above. Generally, the specimen slide on which the specimen is placed may be immersed in one or more reagents corresponding to the immunostaining method under appropriate temperature and time conditions (for example, overnight at 4 ° C.). . Various reagents necessary for immunostaining, ie, fluorescent-labeled primary / secondary antibody, biotin-modified primary / secondary antibody, avidin-modified secondary antibody / fluorescent nanoparticles, etc. are dissolved, and blocking of BSA or the like is performed as necessary. A solution such as a buffer to which the agent has been added can be prepared according to a known method, and can also be obtained as a commercial product.

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

<Staining treatment for cell morphology observation in the first embodiment>
In the staining step of the first embodiment of the present invention, after labeling the specimen slide with fluorescent nanoparticles as a labeling substance, the specimen slide is stained with a morphological observation staining solution in order to obtain information on the shape of the cell or tissue and the position of each part of the cell. Can be performed. Examples of the morphological observation staining solution include a hematoxylin staining solution, an eosin staining solution, and a Papanicolaou (Pap) staining solution.

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

  In addition to HE staining, for example, as described in International Publication WO2015 / 146896, a biological material different from a target biological material constantly expressed in a cell membrane of a tissue section (hereinafter, referred to as a reference biological material) In particular, by fluorescently labeling proteins (antigens) with fluorescent nanoparticles, an image of a cell membrane (cell membrane region) can be formed, and the shape of a cell or tissue and positional information of each part of a cell can be obtained. . In the present invention, such a process of performing fluorescent labeling with a reference biological substance and a solution used for the process are also considered to be an embodiment of a staining process and a staining solution.

  It is appropriate that the reference biological material is selected from biological materials whose expression level does not fluctuate greatly even in a specific disease, unlike the target biological material. For example, membrane proteins such as ATPase, cadherin, cytokeratin, EpCAM (Epithelial Cell Adhesion / Activating Molecule: also referred to as epithelial cell adhesion molecule, CD326, KSA or TROP1) can be preferred reference biological materials in the present invention.

  The reference biological material may be one type, but preferably two or more types. By using two or more types of reference biomaterials, it is possible to further suppress the fluctuation of the total amount of expression and to draw out the outline of the cell membrane with a large number of fluorescent labels, so that the stability as a reference biomaterial can be improved. Performance can be improved.

  The staining agent for fluorescently labeling the reference biological substance by immunostaining includes at least a substance that specifically binds to the reference biological substance, and fluorescent nanoparticles.

  As the substance that specifically binds to the reference biological substance, an antibody (IgG) that specifically recognizes and binds to a protein as a reference biological substance as an antigen as described above can be used. For example, when cadherin is used as the target biological substance, an anti-cadherin antibody can be used as the second probe.

<Fluorescent labeling (FISH)>
The fluorescent labeling process performed in the second embodiment of the present invention is a process of labeling a target gene with fluorescent nanoparticles based on the FISH method in the staining step.

There are various methods in the FISH method, and there is no particular limitation as long as a specimen can be stained so that a target gene can be fluorescently labeled and used for pathological diagnosis or the like. Something like:
A method of preparing a fluorescent-labeled probe in which a fluorescent substance and a probe are linked, and directly labeling and staining a target gene with the fluorescent-labeled probe (direct method);
A biotin-modified probe in which a probe and biotin are linked, and an avidin-modified fluorescent nanoparticle in which a fluorescent substance is linked to avidin or streptavidin are prepared.After the biotin-modified probe is reacted with the target gene, the avidin-modified fluorescent nanoparticle is further reacted. Then, the target gene is indirectly fluorescently labeled and stained using an avidin-biotin reaction (indirect method).

  In the above-mentioned indirect method, hapten (a substance having a relatively low molecular weight which has no immunogenicity but shows antigenicity and can react with an antibody) and an anti-hapten antibody such as dicoxigenin and anti-hapten are used in place of biotin and avidin. Combinations of dicoxigenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antigens, as well as other substances with similar specific reactivity, can also be utilized.

  The FISH method may be performed according to standard procedures and processing conditions for each of the various methods described above. In general, a specimen slide in which a specimen is mounted on a slide may be immersed in one or two or more reagents according to the FISH method under appropriate temperature and time conditions. Various reagents necessary for FISH, that is, a solution such as a buffer solution in which a fluorescent labeling probe, a biotin-modified probe, an avidin-modified fluorescent substance, and the like are dissolved, and a blocking agent such as BSA is added as necessary, are prepared by a known method. Therefore, it can be produced and can be obtained as a commercial product. For example, by replacing the dTTP of the DNA clone of the target gene with the biotin-modified dUTP by the nick translation method, a biotin-modified probe in which a plurality of biotins have been introduced into the DNA probe can be produced.

  The target gene to be subjected to FISH is not particularly limited, but typically, a protein targeted for pathological diagnosis based on the FISH method, for example, HER2, TOP2A, HER3, EGFR, P53, MET, ki67 , And various other cancer / tumor-related genes (so-called biomarker genes), as well as cancer-related genes such as cancer growth factors, transcription factors, growth factor receptors, and transcription factor receptors. You can choose.

  A probe for the target gene can be prepared according to a known method, and can also be obtained as a commercial product. The nucleotide length, nucleotide sequence, and GC content of the probe can be adjusted to have appropriate stringency in consideration of hybridization conditions.

<Staining treatment for cell morphology observation in the second embodiment>
The morphological observation staining process mainly performed in the second embodiment of the present invention is a process of performing nuclear staining with an aqueous staining reagent (a fluorescent dye having a nuclear staining ability used in the form of an aqueous solution) in a staining process. By performing the nuclear staining treatment, the number of cells can be counted, and the number of bright spots of fluorescent nanoparticles that have fluorescently labeled the target gene in the nucleus (on the chromosome) can be counted.

  Representative aqueous dyes for the nucleus include, but are not limited to, DAPI (4,6-diamidino-2-phenylindole dihydrochloride), a fluorescent dye that intercalates into double-stranded DNA is not. Nuclear staining using an aqueous staining reagent can be performed according to a conventional method.

<Fluorescent nanoparticle fixing treatment>
The fluorescent nanoparticle fixing treatment performed in the first embodiment and the second embodiment of the present invention is an immunostaining step in the first embodiment, and a FISH staining step in the second embodiment. This is a process performed before the morphological observation staining process to be performed, in which the fluorescent nanoparticles introduced into the tissue section are fixed using a predetermined fixing reagent.

  In the first embodiment, a fluorescent labeling process based on an immunostaining method for a protein (antigen) as a target biological material is performed, and then in a second embodiment, a fluorescent labeling process based on a FISH method for a nucleic acid (gene) as a target biological material. After performing the above, the fluorescent nanoparticles fixed to the target biological material of the tissue section are cross-linked with the protein around it by performing the fluorescent nanoparticle fixing treatment, or the area around the tissue section to which the fluorescent nanoparticles are bonded is Proteins can be crosslinked. Therefore, even if the morphological observation staining treatment and / or the washing treatment or the blocking treatment is performed thereafter, the fluorescent nanoparticles labeled with the target biological substance are dissociated or released from the vicinity of the target biological substance (for example, in the second embodiment). It is presumed that this can prevent out of the nucleus) and solve the problem that the number of fluorescent luminescent spots of the fluorescent nanoparticles decreases.

(Immobilized reagent)
The immobilized reagent for fluorescent nanoparticles used in the present invention includes a reaction site (hereinafter, referred to as “first reaction site”) present on the surface of the fluorescent nanoparticle and a reaction site (hereinafter, referred to as “first reaction site”) of a protein around a tissue section. This is a compound (crosslinking agent) having a reaction site (hereinafter, referred to as a “third reaction site” and a “fourth reaction site”, respectively) capable of reacting with each of the “second reaction sites”.

  Examples of the first reaction site and the second reaction site include, for example, an amino group (ε-amino group of a lysine side chain, an α-amino group at the N-terminus), a sulfhydride group (thiol group), a hydroxy group, a carboxy group, an aromatic ring and the like. Functional groups. The first reaction site further includes a site that specifically binds to a specific molecule, such as streptavidin and avidin. The first reaction site and the second reaction site may be the same or different.

  On the other hand, examples of the third reaction site and the fourth reaction site include functional groups such as an NHS (N-hydroxysuccinimide) group, a maleimide group, an aldehyde group, an epoxy group, and an iodine group. Examples of the third reaction site further include biotin specifically binding to streptavidin, avidin and the like. The third reaction site and the fourth reaction site may be the same or different, and one functional group may have both functions of the third reaction site and the fourth reaction site ( For example, an aldehyde group when formaldehyde is used as an immobilizing reagent). The third and fourth reaction sites may be linked by a linker such as PEG. A plurality of third and / or fourth reaction sites may be present in one molecule of the immobilized reagent.

  Specific examples of the immobilizing reagent (crosslinking agent) include aldehyde-based crosslinking agents such as paraformaldehyde (4% paraformaldehyde aqueous solution), formaldehyde (20% formalin), and glutaraldehyde; Bi-Functional PEG DE34HS, Bi-Functional PEG DE100HS. , Bi-Functional PEG DE200HS, 4-arm-Functional PEG PTE-100HS, 4-arm-Functional PEG PTE-200HS, 4-arm-Functional PEG PTE-400HS, 8-arm-Function-G10E -Functional HGEO PTE-200GS, 8-arm-Funct NHS ester-based cross-linking agents such as onal HGEO PTE-400GS; Bi-Functional PEG DE 100MA, Bi-Functional PEG DE 200MA, Bi-Functional PEG DE 400MA, 4-arm-Functional PEG PTE-OnMPEG Maleimide crosslinking agents such as PTE-200MA, 4-arm-Functional PEG PTE-400MA; N-hydroxysuccinimide (NHS) groups such as NHS-PEG12-MAL, NHS-PEG1000-MAL, NHS-PEG5000-MAL and maleimide ( (MAL) group cross-linking agent linked via a linker; epoxy-PEG-epoxy (Mw: 500) Epoxy -PEG- Epoxy (Mw: 2000) epoxy crosslinking agents such as.

  Among the immobilization reagents as described above, the immobilization reagent having an NHS group and / or an epoxy group as a functional group of the third reaction site and / or the fourth reaction site is the first reaction site and / or the epoxy group of the fluorescent nanoparticle surface. Alternatively, it is preferable because it easily forms a bond with the second reaction site of the protein in the tissue section. In particular, since the immobilization reagent containing an NHS group is sufficiently crosslinked even at a low concentration, the target biological substance (and the reference biological substance) on the tissue section and the protein used for the crosslinking are less likely to undergo structural change. Therefore, it is preferable. In the first embodiment, further, from the viewpoint that the binding force of the antigen-antibody reaction in the fluorescent labeling treatment (and the staining treatment for the reference biological substance) can be kept high by not causing the structural change of the protein. preferable.

  In addition, the immobilization reagent to which the functional group is bonded via a relatively long linker such as PEG can be used as the first reaction site on the surface of the fluorescent nanoparticle and the second reaction site on the protein of the tissue slice due to the flexible movement of the linker. Not only is it easy to cause a cross-linking reaction with the linker, but also it is presumed that the linkers cover each other in a mesh form, which can further strengthen the bond between the fluorescent nanoparticle and the protein of the tissue section.

  The fluorescent nanoparticle fixing treatment can be performed by bringing an appropriate concentration of an immobilizing reagent into contact with a stained slide for an appropriate time. The concentration of the immobilization reagent in the fixing treatment solution can be appropriately adjusted, but is preferably, for example, 1 to 500 μM. In particular, when using an immobilization reagent having a functional group bonded to both ends via a linker such as PEG, if the concentration is too high, the crosslinking reaction does not proceed sufficiently. It binds to the section or fluorescent nanoparticle, and the other functional group on the other side via the linker remains unreacted without reacting with the cross-linking partner. (Combined). In addition, if the fluorescent nanoparticles and the tissue section are covered with an excess of the immobilizing reagent, the intensity of the fluorescent light emitted from the fluorescent nanoparticles decreases, making it difficult to distinguish the fluorescent nanoparticles and the tissue sections. The reaction between the solution and the target may be hindered, and the staining by those treatments may be insufficient. The contact time between the immobilization solution and the cells (immobilization time) can also be appropriately adjusted, and is, for example, about several minutes to several hours at room temperature.

(Cell fixation treatment)
In the first embodiment and the second embodiment of the present invention, the cell fixing treatment performed as necessary is performed in the specimen pretreatment step of each embodiment, for example, in the first embodiment, after the antigen activation treatment, In the embodiment, the treatment is performed after the enzyme (protease) treatment to suppress the detachment of the cells from the preparation. In the present invention, the cell fixing treatment is distinguished from the fluorescent nanoparticle fixing treatment in that the cell fixing treatment is performed in the specimen pretreatment step (that is, before the staining step). In the cell fixing treatment, for example, after a sample slide is immersed in a formalin solution for a certain period of time, the sample slide is immersed in a washing buffer for washing, and this operation is repeated a plurality of times as necessary, and then the sample slide is dried by air drying or the like It is sufficient to do so.

(Blocking processing)
In the present invention, the blocking treatment performed as necessary is performed before and after the antigen-antibody reaction or the biotin-avidin reaction in the immunostaining treatment (first embodiment) or the FISH treatment (second embodiment) performed in the staining step. This is a process for suppressing nonspecific adsorption of antibodies or adsorption of avidin to endogenous biotin.

  In humans, endogenous biotin is abundant in the liver, kidney, muscle, mammary gland, and digestive tract in humans, and is used for cryopreservation of tissue sections and fixation (preparation of immobilization reagent used in the sample preparation process). It may not be deactivated depending on conditions such as type. Therefore, when a section derived from the above tissue such as a mammary gland is to be stained, it is preferable to perform a blocking treatment for a biotin-avidin reaction.

  For example, in the above-described secondary antibody method or the avidin-biotin combined secondary antibody method in the first embodiment, before the treatment for binding the primary antibody to the target biological substance, In the case where membrane staining using a reference biological substance is performed as a morphological observation staining treatment before the treatment for binding the labeled or biotin-modified secondary antibody, the primary antibody is bound to the reference biological substance. Before the treatment, or before the treatment of binding the primary antibody to a fluorescently labeled or biotin-modified secondary antibody, the primary antibody or the secondary antibody targets non-specific proteins other than the predetermined antigen. A blocking treatment for an antigen-antibody reaction for suppressing specific adsorption is performed.

  In the first embodiment, the avidin-biotin combined primary antibody method or the avidin-biotin combined secondary antibody method described above, before the treatment of binding the avidin-modified fluorescent nanoparticles to the biotin-modified primary antibody, Before the treatment of binding the avidin-modified fluorescent nanoparticle to the biotin-modified secondary antibody, for example, to prevent the avidin-modified fluorescent nanoparticle from adsorbing on endogenous biotin instead of the predetermined biotin to be targeted And a blocking treatment for avidin-biotin reaction is performed.

  On the other hand, also in the indirect method described in the second embodiment, a fluorescent-labeled anti-hapten antibody (eg, an anti-digoxigenin antibody), a biotin-modified anti-hapten antibody, or an unmodified antibody is used for a hapten (eg, digoxigenin) -modified probe bound to a target gene. Before the treatment for binding the anti-hapten antibody (primary antibody), an antibody against a fluorescently labeled or biotin-modified anti-hapten antibody (secondary antibody, for example, anti-mouse IgG antibody) is added to the unmodified anti-hapten antibody. Before the binding treatment, a blocking treatment for an antigen-antibody reaction may be performed.

  In the indirect method of the second embodiment, when an avidin-biotin reaction is used in combination, avidin modification is applied to a biotin-modified probe bound to a target gene or a biotin-modified anti-hapten (primary or secondary) antibody. Before the treatment for binding the fluorescent nanoparticles, a blocking treatment for avidin-biotin reaction may be performed.

  Here, in the case where the membrane staining using the reference biological substance is performed in the first embodiment (secondary antibody method) as described above, after the treatment using the fluorescently labeled secondary antibody for the target biological substance is completed, In some cases, a blocking treatment is performed for the substance using a fluorescently labeled secondary antibody (for example, a blocking agent is added to a solution of the fluorescently labeled secondary antibody). By performing the fluorescent nanoparticle fixing treatment between the treatment using the secondary antibody and the treatment using the fluorescently labeled secondary antibody for the reference biological substance, the treatment using the fluorescently labeled secondary antibody for the reference biological substance is further performed. By performing the fluorescent nanoparticle fixing treatment after completing the above, it is possible to suppress the dissociation of the fluorescent nanoparticles contained in each fluorescently labeled secondary antibody.

  Similarly, when a staining process is performed on a plurality of target biological substances (antigen in the first embodiment, genes in the second embodiment), a fluorescent-labeled secondary antibody or avidin-modified fluorescent nanoparticle for the first target biological substance is also used. Between the fluorescent labeling process using particles or the like and the fluorescent labeling process on the second target biological material, if necessary, after finishing the fluorescent labeling process on the second target biological material (third target biological material) By performing the fluorescent nanoparticle fixing treatment (before starting the fluorescent labeling treatment on the substance), it is possible to suppress the dissociation of the fluorescent nanoparticles by the blocking treatment for the antigen-antibody reaction or the avidin-biotin reaction.

  The blocking treatment may be performed using a known blocking agent. For example, BSA-containing PBS buffer and the like can be used for the blocking treatment for the antigen-antibody reaction, and the avidin solution (the treatment for blocking by binding to endogenous biotin) can be used for the blocking treatment for the avidin-biotin reaction. Liquid) and a biotin solution (to bind to unreacted sites among the four reactive sites of the avidin and seal them, so that avidin-modified antibodies or fluorescent nanoparticles used in the present invention do not bind) ) (For example, Nichirei Biosciences Inc. “Endogenous Avidin / Biotin Blocking Kit”). As described above, when a single sample slide (tissue section) is subjected to a plurality of staining treatments (antigen-antibody reaction or avidin-biotin reaction), if necessary, a blocking treatment and a fluorescent nanoparticle prior thereto may be performed. The fixing process may be performed a plurality of times.

<Sample post-processing step>
The specimen slide that has been subjected to the staining steps of the first embodiment and the second embodiment of the present invention can be subjected to an encapsulation process so as to be suitable for observation. If necessary, in the first embodiment, it is preferable to perform the clearing and dehydrating treatment, and in the second embodiment, it is preferable to perform the solvent replacement and the dehydrating treatment. Conditions for performing these treatments, for example, the temperature and immersion time when immersing the sample slide in a predetermined treatment solution can be appropriately adjusted according to a conventional immunostaining method so that an appropriate signal is obtained. it can.

[Preparation Example 1: SA-Modified Fluorescent Dye-Integrated Nanoparticle 1] Preparation of Streptavidin-Modified Fluorescent Dye-Integrated Silica Particles (Average Particle Diameter: 160 nm)
3.4 mg of Texas red dye molecule “Sulfforhodamine 101” (Sigma-Aldrich) used for fluorescent labeling and 3 μL of 3-aminopropyltrimethoxysilane (KBM903, manufactured by Shin-Etsu Silicone Co., Ltd.) in N, N-dimethylformamide (DMF) To obtain an organoalkoxysilane compound.

  0.6 mL of the obtained organoalkoxysilane compound was mixed with 48 mL of 99% ethanol, 0.6 mL of tetraethoxysilane (TEOS), 2 mL of ultrapure water, and 2.0 mL of 28% by mass ammonia water at 5 ° C. for 3 hours. .

  The mixture prepared in the above step was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. Ethanol was added to the precipitate to disperse the precipitate, followed by rinsing for centrifugation again. Furthermore, the same rinsing was repeated twice to obtain TexasRed dye-integrated silica nanoparticles. SEM observation was performed on 1000 of the obtained nanoparticles, and the average particle diameter was measured as described above. As a result, the average particle diameter was 160 nm.

The obtained fluorescent dye-incorporated nanoparticles are adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM EDTA (ethylenediaminetetraacetic acid), and the solution is adjusted to a final concentration of 10 mM. SM (PEG) ₁₂ (succinimidyl-[(N-maleimidopropionamide) -dodecaneethylene glycol] ester, manufactured by Thermo Scientific) was mixed and reacted at 5 ° C. for 1 hour.

This mixture was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. Then, PBS containing 2 mM of EDTA was added to disperse the precipitate, followed by centrifugation again. By performing washing in the same manner three times, fluorescent dye-integrated silica particles having a maleimide group at the end were obtained.
Streptavidin capable of binding to fluorescent dye-integrated silica particles was prepared as follows.

First, after adding 40 μL of streptavidin (manufactured by Wako Pure Chemical Industries) adjusted to 1 mg / mL to 210 μL of borate buffer, 70 μL of 2-iminothiolane hydrochloride (manufactured by Sigma-Aldrich) adjusted to 64 mg / mL was added. The reaction was performed at room temperature for 1 hour. Thus, a thiol group to the amino group of streptavidin _{(-NH-C (= NH 2} + Cl -) -CH 2 -CH 2 -CH 2 -SH) was introduced.

  The streptavidin solution was desalted with a gel filtration column (Zaba Spin Destoring Columns: Funakoshi) to obtain streptavidin capable of binding to the silica particles. This total amount of streptavidin (containing 0.04 mg) was mixed with 740 μL of the above silica particles adjusted to 0.67 nM using PBS containing 2 mM of EDTA, and reacted at room temperature for 1 hour.

  The reaction was stopped by adding 10 mM mercaptoethanol. After concentrating the obtained solution with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification, to obtain streptavidin-modified Texas Red-integrated silica nanoparticles (SA-modified fluorescent dye-incorporated nanoparticles 1).

[Preparation Example 2: SA-Modified Fluorescent Dye-Integrated Nanoparticles 2] Preparation of Streptavidin-Modified Fluorescent Dye-Integrated Silica Particles (Average Particle Diameter: 320 nm)
Streptavidin-modified Texas Red dye-integrated silica particles (SA-modified fluorescent dye-integrated nanoparticles 2) were prepared in the same manner as in Production Example 1 except that the amount of use of 28% by mass of aqueous ammonia was changed from 2.0 mL to 2.6 mL. Manufactured.

[Preparation Example 3: SA-Modified Fluorescent Dye-Integrated Nanoparticles 3] Preparation of Streptavidin-Modified Fluorescent Dye-Integrated Silica Particles (Average Particle Size: 280 nm)
Streptavidin-modified Texas Red dye-integrated silica particles having an average particle size of 280 nm (SA-modified fluorescent dye) in the same manner as in Production Example 1 except that the amount of use of 28% by mass of aqueous ammonia was changed from 2.0 mL to 2.5 mL. Production of integrated nanoparticles 3) was performed.

[Preparation Example 4: SA-Modified Fluorescent Dye-Integrated Nanoparticle 4] Preparation of Streptavidin-Modified Fluorescent Dye-Integrated Silica Particles (Average Particle Diameter: 80 nm)
Streptavidin-modified Texas Red dye-integrated silica particles (SA-modified fluorescent dye-integrated nanoparticles 4) were prepared in the same manner as in Production Example 1 except that the amount of use of 28% by mass of aqueous ammonia was changed from 2.0 mL to 1.7 mL. Manufactured.

[Preparation Example 5: SA-Modified Fluorescent Dye-Integrated Nanoparticles 5] Preparation of Streptavidin-Modified Fluorescent Dye-Integrated Silica Particles (Average Particle Diameter: 40 nm)
Streptavidin-modified Texas Red dye-integrated silica particles (SA-modified fluorescent dye-integrated nanoparticles 5) were prepared in the same manner as in Production Example 1 except that the amount of use of 28% by mass of aqueous ammonia was changed from 2.0 mL to 1.2 mL. Manufactured.

[Preparation Example 6: SA-Modified Fluorescent Dye-Integrated Nanoparticles 6] Preparation of Streptavidin-Modified Fluorescent Dye-Integrated Melamine Resin Particles (Average Particle Diameter: 155 nm)
After dissolving 2.5 mg of Texas Red dye molecule “Sulforhodamine 101” (Sigma-Aldrich) in 22.5 mL of pure water, the mixture was stirred for 20 minutes while maintaining the temperature of the solution at 70 ° C. with a hot stirrer. To the solution after stirring, 1.5 g of a melamine resin "Nikarac MX-035" (Nippon Carbide Industry Co., Ltd.) was added, and the mixture was further heated and stirred for 5 minutes under the same conditions. 100 μL of formic acid was added to the solution after stirring, and the solution was stirred for 20 minutes while maintaining the temperature of the solution at 60 ° C. Then, the solution was allowed to cool to room temperature. The cooled solution was dispensed into a plurality of centrifuge tubes and centrifuged at 12,000 rpm for 20 minutes to precipitate fluorescent dye-incorporated melamine resin particles contained in the solution as a mixture. The supernatant was removed, and the precipitated resin particles were washed with ethanol and water.

0.1 mg of the washed resin particles are dispersed in 1.5 mL of ethanol, 2 μL of aminopropyltrimethoxysilane “LS-3150” (Shin-Etsu Chemical Co., Ltd.) is added, and the mixture is reacted at room temperature for 8 hours with stirring. A surface amination treatment was performed. The concentration of the resin particles whose surface was aminated was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and a linker reagent “SM (PEG) ₁₂ ” (manufactured by Thermo Scientific) was added to this solution. , Cat. No. 22112) was added to a final concentration of 10 mM, mixed, and reacted at room temperature for 1 hour with stirring. The reaction solution was centrifuged at 10,000 G for 20 minutes, and the supernatant was removed. Then, PBS containing 2 mM of EDTA was added to disperse the precipitate, and the mixture was centrifuged again under the same conditions. By performing washing by the same procedure three times, resin particles surface-modified with a PEG chain having a maleimide group at the end were obtained.

On the other hand, streptavidin into which a thiol group was introduced was prepared as follows. First, 70 μL of an aqueous solution of N-succinimidyl-S-acetylthioacetate (SATA, product of Pirate) adjusted to 64 mg / mL was added to 40 μL of an aqueous solution of streptavidin (Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg / mL. , by reacting from 1 hour at room temperature, it was introduced protected thiol group to the amino group of streptavidin _{(-NH-CO-CH 2 -S} -CO-CH 3). Subsequently, a free thiol group (-SH) was generated from the protected thiol group by hydroxylamine treatment, and the treatment of introducing the thiol group (-SH) into streptavidin was completed. The solution was desalted through a gel filtration column (Zaba Spin Desalting Columns: Funakoshi) to obtain streptavidin into which a thiol group was introduced.

  The prepared fluorescent dye-incorporated melamine resin particles surface-modified with a PEG chain having a maleimide group at the end and the prepared thiol group-introduced streptavidin are mixed in PBS containing 2 mM of EDTA and reacted for 1 hour. Thus, streptavidin was bound to the resin particles via the PEG chain. 10 mM mercaptoethanol was added to the reaction solution to stop the reaction. After concentrating the obtained solution with a centrifugal filter, unreacted substances were removed using a gel filtration column for purification to obtain streptavidin-modified fluorescent dye-loaded melamine resin particles (SA-modified fluorescent dye-loaded nanoparticles 6).

[Preparation Example 7] Preparation of biotin-modified BAC probe for HER2 gene CellBiochemBiophys. 2006; 45 (1) 59, a biotin-modified BAC probe was prepared by the nick translation method. That is, a kit for nick translation (product name “GSP-Nick Translation Kit” K-015, manufactured by GSP) was used for 1 μg (5 μL) of HER2-DNA clone (about 150 kbp) purchased from GSP. And the dTTP of the HER2-DNA clone (nucleic acid molecule) was replaced with biotin-modified dUTP. The specific manufacturing procedure is as follows.

First, the following reagents were mixed in a centrifuge tube.
10 × NickBuffer (Tris-HCl [pH 7.2], MgSO ₄ , DTT): 2.5 μL,
・ BSA (Nuclease-free BSA): 1.5 μL
・ DNTP mix (dATP, dCTP, dCTP): 5 μL
・ DTTP: 1.5 μL
・ Biotin-16-dUTP (product number 1093070, manufactured by Roche, 50 nmol / 50 μL): 0.2 μL
・ Pure water (Nucleate free water) ・ ・ ・ 3μL
・ The above HER2-DNA clone (about 150 kbp) 1 μg aqueous solution 5 μL
-DNA polymerase I (Tris-HCl [pH 7.5], EDTA, DTT, glycerol): 1 μL
・ DNaseI: 5 μL

  Next, the mixed reagent was reacted at 15 ° C. for 4 hours, and heated at 70 ° C. for 10 minutes to stop the reaction. After the reaction, 25 μL of distilled water was added to the centrifuge tube. The reaction solution of the biotin-labeled BAC probe was purified using a microspin column for nucleic acid purification (“MicroSpin S-200HR Column” manufactured by GE Healthcare, product number “# 27-5120-01”).

  About 5.56 μL of a 3 M sodium acetate solution (pH 5.2) and 150 μL of 100% ethanol were added to this solution, and the mixture was allowed to stand at −20 ° C. for 1 hour or more. A precipitate was formed by centrifugation at 16000 rpm for 10 minutes at 4 ° C. Further, 500 μL of 70% ethanol was added, and the mixture was centrifuged at 4 ° C. and 16,000 rpm for 1 minute to remove the supernatant. 5-10 μL of distilled water was added to the precipitate to completely dissolve it, and a solution of a biotin-modified BAC probe having a final concentration of 1 μg / 250 μL was obtained.

[Preparation Example 8] Preparation of fluorescent-labeled probe for HER2 gene 25 μL (concentration: 1 μg / 250 μL) of biotin-labeled BAC probe prepared in Preparation Example 7 and streptavidin-modified fluorescent dye integrated in Preparation Example 6 A solution of melamine resin particles (SA fluorescent dye-integrated nanoparticles 6) is mixed and reacted at room temperature for 30 minutes to bind biotin and streptavidin, respectively, to obtain a DNA probe for HER-2 detection. Was.

  The fluorescence-labeled DNA probe obtained as described above was combined with a hybridization buffer (25% deionized formamide, 2 × SSC, 200 ng / μL salmon sperm DNA, 5 × Denhardt's solution, 50 mM sodium phosphate, pH 7.0, (1 mM EDTA) to a final concentration of 1 to 5 ng / μL. Free ligand (free streptavidin, biotin, and dATP substrates) was removed by a S300 size spin column (Amersham Biosciences, Piscataway, NJ). This fluorescently labeled DNA probe was stored frozen at -20 ° C until use.

[Preparation Example 9] Preparation of biotin-modified trastuzumab As trastuzumab, powdered Herceptin (registered trademark) manufactured by Roche in the form of a pharmaceutical was used, and Biotin Labeling kit-SH (Dojindo Chemical, The biotin-modified trastuzumab was obtained by performing a biotin modification using CORD LK10).

[Preparation Example 10] Preparation of biotin-modified anti-ki67 antibody An anti-ki67 antibody (clone SP6, Abcam cord: ab16667) was used as a primary antibody, and a Biotin Labeling kit-SH (Dojindo, cord LK10) was used for this. By performing biotin modification, a biotin-modified ki67 antibody was obtained.

[Preparation Example 11] Preparation of biotin-modified anti-rabbit IgG antibody In a 50 mM Tris solution, 50 µg of anti-rabbit IgG antibody (LO-RG1, GeneTex, cordGTX40383) used as a secondary antibody was dissolved. A DTT (dithiothreitol) solution was added to this solution to a final concentration of 3 mM, mixed, and reacted at 37 ° C. for 30 minutes. Thereafter, the reaction solution was passed through a desalting column "Zeba Desalt Spin Columns" (Thermo Scientific, Cat. # 89882) to purify the secondary antibody reduced with DTT. 200 μL of the total amount of the purified antibody was dissolved in a 50 mM Tris solution to prepare an antibody solution. On the other hand, the linker reagent "maleimide-PEG ₂ - biotin" (Thermo Scientific Corporation, Product No. 21901) was adjusted to 0.4mM with DMSO. 8.5 μL of this linker reagent solution was added to the antibody solution, mixed, and reacted at 37 ° C. for 30 minutes, thereby binding biotin to the anti-rabbit IgG antibody via a PEG chain. The reaction solution was purified by passing through a desalting column. The concentration of the protein (biotin-modified secondary antibody) in the reaction solution was calculated by measuring the absorbance of the desalted reaction solution at a wavelength of 300 nm using a spectrophotometer (Hitachi "F-7000"). A solution in which the concentration of the biotin-modified secondary antibody was adjusted to 250 μg / mL using a 50 mM Tris solution was used as a biotin-modified secondary antibody solution.

-I. Embodiment based on immunostaining method (first embodiment)-
[Comparison with and without fluorescent nanoparticle fixation treatment]
In applying the immunostaining and the morphological staining to the same tissue section, during the staining process (when the morphological observation stain is HE staining) or after both staining processes are completed (the morphological staining is the reference biological section). The effect of the presence or absence of the fluorescent nanoparticle fixing treatment performed on the substance (in the case of immunostaining of cadherin) was evaluated by the following method.

[Example 1]
A sample pretreatment step (deparaffinization treatment, activation treatment), a staining step (primary antibody treatment, secondary antibody treatment, fluorescent labeling treatment, fluorescent nanoparticle fixation) are performed according to the following procedures (1) to (3). Processing and staining for morphological observation), and post-processing of the specimen (washing and encapsulating), a stained slide based on the immunostaining method was prepared. Using the prepared stained slide, observation and imaging were performed according to the procedure shown in (4) below.

(1) Specimen pre-treatment step (1-1) Deparaffin treatment Tissues of Cosmo Bio Inc. which have previously calculated the FISH score using a path vision HER-2 DNA probe kit (Abbott) as a specimen slide of a HER2 positive staining control specimen. An array slide (CB-A712) was used. The deparaffinization treatment was performed according to the following procedures (i) to (iii). (I) A sample slide is immersed in a container containing xylene at room temperature for 30 minutes. Xylene was replaced three times during the process. (Ii) Immerse the sample slide in a container containing ethanol at room temperature for 30 minutes. On the way, the ethanol was changed three times. (Iii) The sample slide was immersed in a container containing water for 30 minutes. On the way, the water was changed three times.

(1-2) Activation treatment After the sample slide was deparaffinized, activation was performed according to the following procedures (i) to (v). (I) Washing was performed by replacing the sample slide with water. (Ii) The sample slide was autoclaved in a 10 mM citrate buffer (pH 6.0) at 121 ° C. for 15 minutes. (Iii) The sample slide after the autoclave treatment was immersed in a container containing PBS for 30 minutes and washed. (Iv) A PBS containing 1% BSA was placed on a sample slide and subjected to a blocking treatment for 1 hour.

(2) Immunostaining step (2-1) Fluorescence labeling treatment After performing the activation treatment (1-2) of the sample slide, it was prepared in Preparation Example 9 diluted to 0.05 nM with 1% BSA-containing PBS buffer. Biotin-modified trastuzumab was reacted with the sample slide for 2 hours, and then washed with PBS. Further, the mixture was reacted with the SA-modified fluorescent dye-integrated nanoparticles 1 prepared in Preparation Example 1 for 0.5 hour, and then washed with PBS.

(2-2) Fluorescent nanoparticle fixation treatment The fluorescent nanoparticle fixation treatment was performed by immersing the sample slide subjected to the fluorescent labeling treatment (2-1) in a 4% neutral paraformaldehyde aqueous buffer solution for 10 minutes. .

(2-3) Morphological Observation Staining Treatment Morphological observation and staining treatment (hematoxylin-eosin staining) is performed on the sample slide that has been subjected to the fluorescent nanoparticle fixing treatment (2-2) by the following procedures (i) to (ii). ) Was done. (I) Hematoxylin staining was performed by staining the sample slide with a Mayer's hematoxylin solution for 5 minutes. Thereafter, the slide was washed with running water at 45 ° C. for 3 minutes. (Ii) Next, the cells were stained with a 1% eosin solution for 5 minutes to carry out eosin staining to prepare stained slides.

(3) Specimen post-processing step After performing the morphological observation staining process (2-3), the staining slide was subjected to the encapsulation process according to the following procedures (i) to (ii). (I) Entelane New (Merck) was dropped on the stained slide at room temperature, covered with a cover glass, and air-dried at room temperature for 10 minutes to perform an encapsulation treatment. (Ii) Thereafter, the stained slide subjected to the encapsulation treatment was stored in a light-shielded state until signal measurement.

(4) Observation The stained slide after the encapsulation treatment was irradiated with predetermined excitation light to emit fluorescence. The stained slide in this state was observed and imaged using a fluorescence microscope (“BX-53” manufactured by Olympus) and a digital camera for microscope (“DP73” manufactured by Olympus). The excitation light was set at 575 to 600 nm by passing through an optical filter. The range of the wavelength (nm) of the fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter. The conditions of the excitation wavelength at the time of microscopic observation and image acquisition were such that the irradiation energy near the center of the visual field was 900 W / cm ² at the excitation of 580 nm. The exposure time at the time of image acquisition was arbitrarily set (for example, set to 4000 μs) so that the luminance of the image was not saturated. The number of bright spots in the HER2 (3+) tissue was an average value of 1000 cells measured by the ImageJ FindMaxims method based on an image taken at 400 times. In addition, fluorescent nanoparticles per cell were calculated from the captured image. FIG. 3A shows the photographed fluorescent image.

[Comparative Example 1] (when the fluorescent nanoparticle fixing treatment is not performed in Example 1)
Preparation and observation of a stained slide were performed in the same manner as in Example 1 except that the fluorescent nanoparticle fixing treatment (2-2) was not performed. FIG. 3B shows the photographed fluorescent image.

[Example 2] (Immunostaining using fluorescent nanoparticles of Preparation Example 6)
In the fluorescent labeling treatment (2-1), preparation and observation of a stained slide were performed in the same manner as in Example 1 except that the SA-modified fluorescent dye-incorporated nanoparticles 6 prepared in Preparation Example 6 were used as the fluorescent nanoparticles. Was.

[Comparative Example 2] (when the fluorescent nanoparticle fixing treatment is not performed in Example 2)
Preparation and observation of a stained slide were performed in the same manner as in Example 2 except that the fluorescent nanoparticle fixing treatment was not performed.

[Example 3] (Immunostaining using ki67 as the target biological substance)
A specimen slide was changed to a paraffin-embedded breast cancer tissue array (product number: BR243) manufactured by US-BioMax, and prepared as a biotin-modified primary antibody in a fluorescent labeling treatment (see the description in (2-1) of Example 1). Preparation and observation of stained slides were performed in the same manner as in Example 2 except that the biotin-modified anti-ki67 antibody prepared in Example 10 was used. The photographed fluorescent image and hematoxylin-eosin stained image are shown in FIG. 4 [A] and FIG. 5 [A].

[Comparative Example 3] (in the case where the fluorescent nanoparticle fixing treatment is not performed in Example 3)
Preparation and observation of a stained slide were performed in the same manner as in Example 3 except that the fluorescent nanoparticle fixing treatment was not performed. The photographed fluorescence image and hematoxylin-eosin stained image are shown in FIGS. 4B and 5B.

[Example 4] (in the case of performing a membrane staining process using a reference biological material)
In order to perform a membrane staining process using a reference biological substance (cadherin) instead of the staining process for morphological observation by HE staining, the immunostaining step (see the description of (2) in Example 1) was changed as follows. Except for this, the preparation and observation of stained slides were performed in the same manner as in Example 3.

  Immunostaining with the biotin-modified antibody for the target biological substance ki67 and membrane staining for the reference biological substance cadherin were performed at once by the following procedures (i) to (vi). (I) Using PBS containing 1% BSA, anti-ki67 antibody (clone SP6, Abcam, cord: ab16667) at a concentration of 0.05 nM and anti-pan cadherin antibody [CH -19] (Abcam, cord: ab6528) at a concentration of 1: 200 was prepared. (Ii) A sample slide subjected to cell fixation treatment was immersed in the reaction solution and reacted at 4 ° C. overnight. (Iii) A solution of the biotin-modified anti-rabbit IgG antibody produced in Production Example 11 was further diluted to 6 μg / mL with PBS containing 1% BSA to prepare a secondary reaction treatment solution of the target biological substance. The sample slide after the primary reaction treatment was washed with PBS, immersed in this secondary reaction treatment solution, and reacted at room temperature for 30 minutes. (Iv) A fluorescent labeling reaction solution was prepared by diluting the SA-modified fluorescent dye-incorporated nanoparticles 6 produced in Preparation Example 6 to 0.02 nM using PBS containing 1% of BSA. The specimen slide after the secondary reaction treatment of the target biological substance was immersed in this fluorescent labeling treatment solution, and reacted at room temperature under a neutral pH environment (pH 6.9 to 7.4) for 3 hours. (V) Alexa Fluor 647-labeled anti-mouse IgG antibody (Life Technology, A21236) diluted 1: 200 using PBS containing 1% BSA to prepare a secondary reaction solution of a reference biological substance. The sample slide after the primary reaction treatment was washed with PBS, immersed in this secondary reaction treatment solution, and reacted at room temperature for 30 minutes. (Vi) Fluorescent nanoparticle fixing treatment was performed by immersing the specimen slides on which the target biological substance and the reference biological substance were subjected to the fluorescent labeling treatment in a 4% neutral paraformaldehyde aqueous buffer solution for 10 minutes.

[Comparative Example 4] (when the fluorescent nanoparticle fixing treatment is not performed in Example 4)
Preparation and observation of a stained slide were performed in the same manner as in Example 4 except that the fluorescent nanoparticle fixing treatment (vi) was not performed.

<Result>
Table 1 shows the results of Examples 1 to 4 and Comparative Examples 1 to 4. For example, in the case of Example 3, the number of bright spots per cell of the target biological material is the number of bright spots located on the cell membrane by superimposing the fluorescence image of FIG. 4A and the HE-stained image of FIG. 5A. (The same applies to other examples and comparative examples in which the drawings are omitted). From the comparison of the results of the corresponding Examples and Comparative Examples, the fluorescent nanoparticles are fixed from the stained slides by performing the morphological observation staining treatment (HE staining or membrane staining with a reference biological substance) by performing the fluorescent nanoparticle fixing treatment. It was found that the dissociation can be suppressed and the staining property when the target biological substance is fluorescently labeled can be maintained, so that the target biological substance can be quantified more accurately than before.

[Comparison by difference in type and concentration of immobilized reagent]
When immunostaining and morphological observation staining were performed on the same tissue section, the effect of the difference in the immobilization reagent (crosslinking agent) for fluorescent nanoparticles was evaluated by the following method.

Example 5 (Fluorescent nanoparticle fixing treatment using NHS ester-based crosslinking agent)
In the fluorescent nanoparticle fixing treatment (see the description of (2-2) in Example 1), instead of 4% neutral paraformaldehyde, an NHS ester-based cross-linking agent “Bi-Functional PEG DE 100HS” (molecular weight 10,000, linear chain) Preparation and observation of stained slides were carried out in the same manner as in Example 2 except that NHS groups were present at both ends of the PEG chain in a state of 700 μM (room temperature, 30 minutes).

[Example 6]
Preparation and observation of stained slides were performed in the same manner as in Example 5 except that the concentration of the crosslinking agent was changed to 500 μM.

[Example 7]
Preparation and observation of stained slides were performed in the same manner as in Example 5, except that the concentration of the crosslinking agent was changed to 50 μM.

Example 8
Preparation and observation of stained slides were performed in the same manner as in Example 5 except that the concentration of the crosslinking agent was changed to 2 μM.

[Example 9] (Immunostaining using NHS ester-based crosslinking agent)
The concentration of the NHS ester-based cross-linking agent is changed from “Bi-Functional PEG DE 100HS” to “4-arm-Functional PEG DE200HS” (molecular weight 10,000, NHS groups are present at a total of four terminals of the branched PEG chain). Preparation and observation of stained slides were performed in the same manner as in Example 5 except that the slides were adjusted to 500 μM and used.

[Example 10]
Preparation and observation of stained slides were performed in the same manner as in Example 9 except that the concentration of the crosslinking agent was changed to 50 μM.

[Example 11] (Immunostaining using NHS ester-based crosslinking agent)
The NHS ester-based cross-linking agent is changed from “4-arm-Functional PEG DE100HS” to “8-arm-Functional PEG HGEO 400GS” (molecular weight: 40,000, and a branched PEG chain having NHS groups at eight terminal ends in total). A stained slide was prepared and observed in the same manner as in Example 9 except that the slide was prepared at a concentration of 500 μM and used.

[Example 12]
Preparation and observation of stained slides were performed in the same manner as in Example 11, except that the concentration of the crosslinking agent was changed to 50 μM.

[Example 13] (Immunostaining using a maleimide-based crosslinking agent)
The immobilization reagent for fluorescent nanoparticles was changed from an NHS ester-based crosslinking agent “Bi-Functional PEG DE 100HS” to a maleimide-based crosslinking agent “Bi-Functional PEG DE100MA” (molecular weight 10,000, maleimide at both ends of a linear PEG chain). Preparation and observation of stained slides were performed in the same manner as in Example 7 except that the group was changed to (existing group).

<Result>
Table 2 shows the results of Examples 5 to 13. Similarly to the case where 4% neutral paraformaldehyde is used, even if the fluorescent nanoparticle fixing treatment is performed using an NHS ester-based crosslinking agent or a maleimide-based crosslinking agent, the fluorescent nanoparticle from the stained slide by the morphological observation staining treatment is obtained. It was found that the dissociation of particles can be suppressed and the staining property when the target biological substance is fluorescently labeled can be maintained, so that the target biological substance (membrane protein) can be quantified more accurately than before.

  Regarding the concentration of the immobilizing reagent for fluorescent nanoparticles, the 4% neutral paraformaldehyde used in the fluorescent nanoparticle immobilization treatment of Examples 1-4 corresponds to a concentration of 13.3 mM. On the other hand, the lowest concentration of the NHS ester-based crosslinking agent or maleimide-based crosslinking agent used in Examples 5 to 13 was 2 μM (0.002 mM), and even the highest concentration was 700 μM (0.7 mM). It can be seen that when an NHS ester-based crosslinking agent or a maleimide-based crosslinking agent is used as an immobilizing reagent, crosslinking can be sufficiently performed even at a concentration lower than 4% neutral paraformaldehyde. In particular, when an NHS ester-based cross-linking agent is used, there is almost no difference in the number of bright spots observed due to the difference in concentration, and sufficient cross-linking can be performed even at a low concentration (rather, the concentration should not be too high). I understand.

[Comparison by difference in average particle size]
When immunostaining and morphological staining were performed on the same tissue section, the effect of the difference in the average particle size of the fluorescent nanoparticles was evaluated by the following method.

[Example 14] (Immunostaining using particles of Preparation Example 2)
In the fluorescent labeling treatment (2-1), preparation and observation of a stained slide were performed in the same manner as in Example 1 except that the SA-modified fluorescent dye-integrated nanoparticles 2 produced in Production Example 2 were used as fluorescent nanoparticles. Was.

[Example 15] (Immunostaining using particles of Preparation Example 3)
In the fluorescent labeling treatment (2-1), preparation and observation of a stained slide were performed in the same manner as in Example 1 except that the SA-modified fluorescent dye-integrated nanoparticles 3 manufactured in Preparation Example 3 were used as fluorescent nanoparticles. Was.

[Example 16] (Immunostaining using particles of Preparation Example 4)
In the fluorescent labeling treatment (2-1), the preparation and observation of the stained slide were performed in the same manner as in Example 1 except that the SA-modified fluorescent dye-integrated nanoparticles 4 manufactured in Preparation Example 4 were used as the fluorescent nanoparticles. Was.

[Example 17] (Immunostaining using particles of Preparation Example 5)
In the fluorescent labeling treatment (2-1), preparation and observation of a stained slide were performed in the same manner as in Example 1 except that the SA-modified fluorescent dye-incorporated nanoparticles 5 produced in Production Example 5 were used as fluorescent nanoparticles. Was.

<Result>
The results of Examples 14 to 17 are shown in Table 3 (for comparison, the results of Example 1 in Table 1 are also shown). Fluorescent dye-integrated nanoparticles having an average particle diameter of 40 nm to 160 nm resulted in an increase in the number of observed bright spots per cell compared to fluorescent dye-integrated nanoparticles having an average particle diameter of 280 nm and 320 nm.

-II. Embodiment based on FISH method (second embodiment)-
[Comparison with and without fluorescent nanoparticle fixation treatment]
In applying FISH staining and nuclear staining for morphological observation to the same tissue section, the effect of the presence or absence of a fixing treatment performed between the staining treatments was evaluated by the following method.

[Example 18]
The copy number of the HER2 gene was measured by FISH. That is, the sample pretreatment step (deparaffin treatment, pretreatment for FISH, enzyme treatment), the staining step (preparation of probe, DNA denaturation treatment, hybridization treatment, By performing post-hybridization washing treatment, fluorescent nanoparticle fixing treatment and nuclear staining treatment with DAPI for morphological observation), and post-sample treatment steps (washing treatment, solvent replacement treatment and encapsulation treatment), a stained slide based on the FISH method can be obtained. Produced. Using the prepared stained slide, observation and imaging were performed according to the procedure shown in (4) below.

(1) Specimen pretreatment step (1-1) Deparaffinized treatment A sample slide of HER2-positive control sample (Roche HER2 Dual ISH 3-in-1 control slide, product code: 109530) was prepared according to the following (i) to (i). The deparaffinization treatment was performed according to the procedure of iv). (I) It is immersed in a deparaffinizing agent "Hemo-De" (Pharma Co., Ltd., main component: d-limonene, antioxidant) at room temperature for 10 minutes. (Ii) Immerse the sample slide in a new “Hemo-De” at room temperature for 10 minutes. Repeat the same operation three times. (Iii) A sample slide is immersed in 100% ethanol at room temperature for 5 minutes, washed twice, and dehydrated. (Iv) Air-dry the specimen slide or dry on a slide warmer at 45-50 ° C.

(1-2) Pretreatment for FISH In order to improve the accessibility of the DNA probe, the cell membrane was subjected to the following procedures (i) to (vi) on the specimen slide on which deparaffinization treatment (1-1) was performed. And the removal process of the protein of a nuclear membrane was performed. (I) Treat the sample slide with 0.2 mol / L HCl at room temperature for 20 minutes. (Ii) Immerse the sample slide in purified water for 3 minutes. (Iii) The sample slide is immersed in a washing buffer (2 × SSC: standard sailine citrate) for 3 minutes. (Vi) The sample slide is immersed in a pretreatment solution (1N NaSCN) at 80 ° C. for 30 minutes. (V) Immerse the sample slide in purified water for 1 minute. (Vi) The sample slide is immersed in a washing buffer (2 × SSC) for 5 minutes, and the same operation is repeated twice.

(1-3) Enzyme treatment The sample slide subjected to the protein removal treatment (1-2) was subjected to the enzyme treatment according to the following procedures (i) to (iv). (I) Take out the pretreated sample slide and attach the lower end of the slide glass to a paper towel to remove excess washing buffer. (Ii) Immerse the sample slide in the protease solution heated to 37 ° C for 10 to 60 minutes. This immersion treatment is performed with 25 mg protease (2500-3000 Units / mg) [pepsin] / 1M NaCl [pH 2.0] in 50 mL at 37 ° C. for 60 minutes to degrade proteins of cell membrane and nuclear membrane, particularly collagen. Processed. (Iii) Immerse the sample slide in the washing buffer for 5 minutes. This operation is repeated twice. (Iv) The sample slide was air-dried.

(1-4) Cell fixation treatment Cell fixation treatment was carried out by reacting 4% paraformaldehyde at room temperature for 10 minutes, and the operation of immersing a sample slide in a washing buffer for 5 minutes was repeated twice to wash.

(2) FISH staining step (2-1) Preparation of probe Return the fluorescently labeled DNA probe solution stored in a frozen state to room temperature, and sufficiently increase the viscosity of the solution to such an extent that a pipetting operation capable of collecting an accurate volume can be performed. And mixed the solution with a vortex mixer.

(2-2) DNA denaturation treatment In order to denature the DNA on the sample slide, the sample slide that has been subjected to the sample pretreatment step (1) is subjected to a DNA denaturation treatment according to the following procedures (i) to (viii). went. (I) Place a wet box (an airtight container, the side of which is taped with a paper towel) on the bottom of a paper towel moistened with water before preparation of the sample slide in a 37 ° C incubator for preliminary use. Heat. (Ii) Check that the pH of the denaturing solution (70% formamide / SSC [150 mM NaCl, 15 mM sodium citrate]) is pH 7.0 to 8.0 at room temperature. The denaturing solution is placed in a coplin jar and warmed in a hot tub until the denaturing solution is 72 ° C. ± 1 ° C. (put in a 72 ± 1 ° C. hot tub for at least 30 minutes). (Iii) Mark with a diamond pen or the like on the back side of the sample slide so that the hybridization area can be identified. (Iv) The sample slide is immersed in a coplin jar containing a denaturing solution at 72 ± 1 ° C. to denature the DNA of the sample slide. (V) Using tweezers, remove the sample slide from the denaturing solution and immediately place it in room temperature 70% ethanol. Shake slide to remove formamide. Immerse the sample slide for 1 minute. (Vi) Remove sample slides from 70% ethanol, place in 85% ethanol and shake slides to remove formamide. Immerse the sample for 1 minute. The same operation is repeated twice with 100% ethanol. (Vii) Attach the lower end of the sample slide glass to a paper towel to remove ethanol, and wipe the back of the slide glass with a paper towel. (Viii) The sample slide was air-dried with a dryer.

(2-3) Fluorescent labeling treatment (hybridization treatment)
The sample slide subjected to the denaturation treatment (2-2) was subjected to a hybridization treatment with a fluorescent-labeled DNA probe by the following procedures (i) to (iii). (I) Add 10 μL of the fluorescence-labeled DNA probe solution onto the sample slide, immediately cover the glass slide with a 22 mm × 22 mm cover glass, and spread the fluorescence-labeled DNA probe solution uniformly while preventing air bubbles from entering. (Ii) Seal the cover glass with a paper bond. (Iii) Put the sample slide in a wet box that has been heated in advance, cover the slide, and perform hybridization for 14 to 18 hours in a 37 ° C. incubator.

(2-4) Post-hybridization washing treatment The post-hybridization washing treatment was performed on the sample slide subjected to the hybridization treatment (2-3) according to the following procedures (i) to (vi). (I) Put post-hybridization wash buffer (2 × SSC / 0.3% NP-40) into coplin jar. Place in a water bath at 72 ° C. ± 1 ° C. for at least 30 minutes and preheat in a water bath until the post-hybridization wash buffer reaches 72 ° C. ± 1 ° C. (Place in a water bath at 72 ° C. ± 1 ° C. for at least 30 minutes) ). (Ii) Prepare another coplin jar containing the post-hybridization wash buffer and maintain at room temperature. (Iii) Remove the paper bond seal with tweezers. (Iv) Place sample slides in post-hybridization wash buffer. Wait for the coverslip to spontaneously peel in the solution. (V) The sample slide was removed from the solution, the excess solution was removed, and the slide was immersed in a post-hybridization wash buffer heated to 72 ± 1 ° C. for 2 minutes. (Vi) Remove the sample slide from the coplin jar and air dry under light protection.

(2-5) Fluorescent nanoparticle fixing treatment An NHS ester-based cross-linking agent “Bi-Functional PEG DE 100HS” (molecular weight 10,000) was prepared by adjusting the sample slide subjected to the post-hybridization washing treatment (2-4) to a concentration of 700 μM. (Room temperature, 30 minutes) to perform a fluorescent nanoparticle fixing treatment.

(2-6) Nuclear Staining Treatment 10 μL of DAPI staining solution (2 μg / mL PBS, Molecular Probes, D1306) was added to the hybridization area of the specimen slide on which the fluorescent nanoparticle fixing treatment (2-5) was performed, After holding at 25 ° C. for 10 minutes, a nuclear staining treatment was performed to prepare a stained slide.

(3) Sample post-treatment step The stained slide was subjected to a washing treatment according to the following procedures (i) to (ii). (I) Enterelane New (Merck) was dropped on the tissue of the stained slide treated with (2). An encapsulation process was performed by covering with a cover glass and air-drying at room temperature for 10 minutes. (Ii) Thereafter, the specimen slide on which the encapsulation treatment was performed was stored under light shielding until signal measurement.

(4) Photographing and Analysis of Fluorescence Image Each of Examples 1 to 3 and Comparative Example 1 as described above using a fluorescence microscope Zeiss imager (Camera: MRm monochrome / with cooling function, objective lens: × 100 oil immersion). Observation of the stained slide prepared in the above and photographing of a fluorescent image (× 1000) were performed. The fluorescent dye (sulforhodamine 101) -integrated melamine resin particles are irradiated with excitation light having an excitation wavelength of 575 to 600 nm using a suitable filter, and fluorescence having a fluorescence wavelength of 610 to 670 nm is observed. I got DAPI irradiated excitation light with an excitation wavelength of 340 to 380 nm using an appropriate filter, observed fluorescence with a fluorescence wavelength of 435 to 485 nm, and acquired the fluorescence image.

[Example 19]
A stained slide was prepared and observed in the same manner as in Example 18, except that the concentration of the crosslinking agent was changed to 500 μM.

[Example 20]
A stained slide was prepared and observed in the same manner as in Example 18, except that the concentration of the crosslinking agent was changed to 50 μM.

[Example 21]
Preparation and observation of stained slides were performed in the same manner as in Example 18, except that the concentration of the crosslinking agent was changed to 2 μM.

[Example 22] (Immunostaining using a maleimide-based crosslinking agent)
The immobilization reagent for fluorescent nanoparticles was changed from an NHS ester-based crosslinking agent “Bi-Functional PEG DE 100HS” to a maleimide-based crosslinking agent “Bi-Functional PEG DE100MA” (molecular weight 10,000, maleimide at both ends of a linear PEG chain). Group was present), and a slide was prepared and observed in the same manner as in Example 18, except that the concentration was changed to 50 μm.

[Comparative Example 5] (without performing fluorescent nanoparticle fixing treatment)
Preparation and observation of a stained slide were performed in the same manner as in Example 18 except that the fluorescent nanoparticle fixing treatment was not performed.

<Result>
Table 4 shows the results of Examples 18 to 22 and Comparative Example 5. Similar to the case based on the immunostaining method, when a stained slide is prepared based on the FISH method, the fluorescent nanoparticle fixing treatment using an NHS ester-based crosslinker or a maleimide-based crosslinker allows the cross-linking thereof to be performed. Even when the concentration of the agent is relatively low, the dissociation of the fluorescent nanoparticles from the stained slide by the morphological observation staining treatment (nuclear staining treatment with DAPI) can be suppressed, and the staining property when the target biological substance is fluorescently labeled is maintained. Therefore, it was found that the target biological substance (gene) can be quantified more accurately than before.

Claims (7)

Processing for labeling target biological substances contained in tissue sections on specimen slides with fluorescent nanoparticles by immunostaining or FISH (fluorescent labeling)
And viewed including processing (fluorescent nanoparticles fixing process) for fixing the tissue sections fluorescent labeling process has been performed by immobilizing reagents for fluorescent nanoparticles,
A method for preventing dissociation of fluorescent nanoparticles from a tissue section, wherein the immobilization reagent is selected from NHS-PEG-NHS, biotin-PEG-NHS and epoxy-PEG-epoxy .   2. The method according to claim 1, further comprising a treatment (staining treatment) and / or a treatment of blocking the tissue section on which the fluorescent nanoparticle fixing treatment has been performed with a staining solution for cell morphology observation. The density of the immobilized reagent is 1Myuemu～500myuM, method according to claim 1 or 2. The method according to claim 2 , wherein the staining solution is selected from hematoxylin, eosin and DAPI. The method according to any one of claims 1 to 4 , wherein the fluorescent nanoparticles are fluorescent dye-integrated nanoparticles or inorganic fluorescent nanoparticle aggregates, and have an average particle diameter of 40 nm to 160 nm. The method according to any one of claims 1, 3, and 5, wherein the fluorescent labeling treatment is performed by a FISH method. 7. The method according to claim 6, further comprising a treatment (staining treatment) of staining the tissue section on which the fluorescent nanoparticle fixing treatment has been performed with a staining solution for cell morphology observation and / or a blocking process, wherein the staining solution is DAPI. The method described in.