JP6524833B2 - Method for encapsulating FISH or immunostaining slide using phosphor-accumulated nanoparticles - Google Patents

Description

  The present invention relates to a method for producing a tissue section slide used for pathological diagnosis and the like. More particularly, the present invention relates to the step of encapsulating a tissue section slide treated by FISH or immunostaining with phosphor-accumulated nanoparticles using an oil-based mounting agent.

  In pathological diagnosis, a tissue section collected from a patient is sliced to prepare a slide, and based on a staining image obtained by staining according to a predetermined method, the morphology of a cell or tissue is observed and expression of a specific biomolecule is obtained. By quantifying and evaluating the level, it is a method of diagnosing various events such as whether the patient suffers from a specific disease or whether a specific therapeutic agent is effective.

  For example, it is a membrane protein produced from HER2 gene (HER2 / neu, c-erbB-2) and / or HER2 gene, which is a type of oncogene, using a tissue section prepared by collecting cancer tissue. By quantifying and evaluating HER2 protein that is presumed to function as a receptor for cancer cell growth factor, the prognosis of breast cancer patients can be diagnosed or molecular targeted therapeutic drug "trastuzumab" (trade name "Herceptin" ( Pathological diagnosis which predicts the therapeutic effect by (trademark) and anti-HER2 monoclonal antibody) is widely performed. In human breast cancer cases, amplification of HER2 gene and overexpression of HER2 protein are observed in 15 to 25%, but overexpression of HER2 in cancer cells basically occurs with gene amplification at the DNA level. Tests for HER2 in cancer tissues are classified into methods for amplification at the DNA level, methods for overexpression at the RNA level, and methods for overexpression at the protein level. Representative methods for testing at the protein level and DNA level are immunostaining or immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Such HER2 test is regarded as clinically important, and standard procedures and criteria (scores) for HER2 test by immunostaining (IHC method) and FISH method respectively are defined by the 2007 ASCO / CAP guidelines. ing.

The FISH method is a method of detecting the copy number of HER2 gene in interphase nuclei of cancer cells on a formalin fixed paraffin embedded tissue section using a fluorescently labeled DNA probe for HER2 gene. Generally, a slide (specimen) carrying a sectioned formalin-fixed paraffin-embedded tissue section is prepared, deparaffinized etc. to make it suitable for hybridization, and then reacted with a fluorescently labeled probe. And hybridize to the intranuclear (on chromosome) HER2 gene. Also, usually, the nucleus (chromosome) is further stained using a fluorescent dye DAPI (4 ', 6-diamidino-2-phenylindole) which intercalates into double-stranded DNA. The tissue sections stained in this manner are subjected to an encasing treatment using a mounting agent containing an antifading agent, and a fluorescence image is taken while irradiating a predetermined excitation light. Then, the captured fluorescence image labeled with the HER2 gene and the fluorescence image of DAPI are superimposed, and the number of bright spots observed in the nucleus (on the chromosome) per cell is measured, and the value is determined according to the value of the HER2 gene. Is determined whether or not it is amplified.
The embodiment of the above FISH method can refer to, for example, Patent Document 1 (Japanese Patent Application Laid-Open No. 2013-046620).

  On the other hand, the immunostaining method (IHC method) is a method for detecting the amount of HER2 protein expressed in the cell membrane on a formalin fixed paraffin embedded tissue section using a fluorescently labeled anti-HER2 antibody. The conventional immunostaining method (the original IHC method) adopted a method (enzyme antibody method) using an anti-HER2 antibody labeled with an enzyme that produces a dye when a predetermined substrate is added. An immunostaining method (fluorescent antibody method) using an anti-HER2 antibody labeled with a fluorescent substance excellent in properties has also been used. As in the case of the FISH method, after preparing slides (specimen) on which sliced formalin-fixed paraffin-embedded tissue sections are placed and performing deparaffinization etc. to make them suitable for immunostaining, , The fluorescently labeled antibody is reacted to bind to the HER2 protein of the cell membrane. The tissue section immunostained in this manner is also subjected to an encasing treatment using a mounting agent containing an antifading agent as in the case of the FISH method, and then a fluorescence image is taken while being irradiated with a predetermined excitation light. . Then, the number of bright spots observed in one cell membrane area per cell is obtained by superimposing the photographed fluorescence image in which the HER2 protein is labeled and the image in which the cell membrane for reference is (fluorescently) stained (fluorescent) Is measured to determine whether or not the HER2 protein is abnormally expressed.

  The embodiment of the above-mentioned immunostaining method (fluorescent antibody method), in particular, a method using phosphor-accumulated nanoparticles as a fluorescent substance, is disclosed, for example, in Patent Document 2 (International Publication WO 2013/035703 Pamphlet), Patent Document 3 (Patent Document 3) International publication WO2013 / 147081 pamphlet), patent document 4 (international publication WO2014 / 136776 pamphlet) etc. can be referred to.

JP, 2013-046620, A International Publication WO2013 / 035703 Brochure International Publication WO2013 / 147081 Brochure International Publication WO 2014/136776 Brochure

  In the conventional FISH method, hybridization is performed using a fluorescently labeled probe, and nuclear staining treatment is performed using an aqueous staining reagent such as DAPI, and then a cover glass using an aqueous sealing agent containing an antifading agent It is normal to perform the encapsulation process below. In some cases, the nuclear staining process and the encapsulating process may be performed simultaneously by using an aqueous solution (such as a buffer solution) in which both the aqueous staining reagent and the aqueous encapsulant are dissolved. However, in the case of such encapsulation treatment, the purpose is particularly when the phosphor-accumulated nanoparticles are rendered hydrophilic by, for example, modification with PEG to prevent nonspecific adsorption or to link a desired reactive group. Notice that there is a problem that the phosphor-accumulating nanoparticles that were hybridized to the gene are released and transferred into the liquid layer remaining between the cover glass and the tissue section, resulting in noise when the fluorescence image is taken The Such a problem is likely to occur if the binding between the target and the nucleic acid of the probe is weak, because the size of the phosphor-accumulated nanoparticles is larger than that of a fluorescent dye or a single phosphor particle (for example, the particle size is about 100 nm). Also, when staining is performed not by FISH but by immunostaining, similarly, there arises a problem that the phosphor-accumulated nanoparticles are liberated during the encapsulation treatment using a water-based encapsulant.

  Therefore, in one aspect of the present invention, when staining is performed by the FISH method or immunostaining method using phosphor-accumulated nanoparticles, the problem of liberation of the phosphor-accumulated nanoparticles in a tissue section slide to be produced is The issue is to eliminate it.

The inventors have further noticed that there is a problem that in such encapsulation treatment, not only the phosphor-accumulated nanoparticles but also DAPI used for nuclear staining may elute out of the nucleus.
Therefore, in another aspect of the present invention, when performing nuclear staining using an aqueous staining reagent such as DAPI, it is an object to solve the problem that the aqueous staining reagent elutes in the prepared tissue section slide. I assume.

  The inventors stain the target biological material by the FISH method or immunostaining method using phosphor-accumulated nanoparticles, and then remove the aqueous solvent such as buffer remaining on the tissue section to remove the oil solvent. It has been found that the release treatment of the phosphor-accumulated nanoparticles can be suppressed by performing an encapsulation treatment using an oil-based encapsulant after substitution with.

  In addition, when replacing the water-based solvent with the oil-based solvent, the inventors may perform air-drying to remove the water-based solvent and then immerse in the oil-based solvent, but the mixed solvent of dehydrated ethanol and xylene may be used. And immersing the tissue section in a mixed solution of an oil-based solvent and an organic solvent having a high affinity (with a degree of affinity for free mixing) that is substantially water-free and has a dewatering action. It was also found that it is preferable to immerse only in an oil-based solvent to complete the solvent substitution treatment after dehydration treatment.

  Furthermore, after performing nuclear staining treatment using DAPI which is a representative nuclear staining agent (water-based staining reagent), the present inventors perform dehydration treatment using a dehydration treatment solution composed of the mixed solvent as described above. Have noticed that DAPI intercalated into DNA may be eluted by ethanol contained in the dehydrating solution. Therefore, prior to the dehydration treatment, the tissue section is immersed in a solvent having high affinity to water containing a solvent that does not elute the aqueous staining reagent used for the nuclear staining treatment, for example, a mixed solvent of PBS in which DAPI is difficult to dissolve is mixed with ethanol. Then, it was also found that the washing treatment can suppress the elution of DAPI in ethanol in the subsequent dehydration treatment, solvent substitution treatment and encapsulation treatment of the tissue surface.

  In Patent Document 3 (International Publication WO2013 / 147081), in immunostaining for a tissue sample, the target protein is labeled with phosphor-accumulated nanoparticles and then "fixed" (for example, in a dilute aqueous paraformaldehyde solution) By immersing the tissue sections immunostained in (e.g.), the problem that the fluorescent label is eluted in the oil-based sealant and the staining ability is lost even if the encapsulating treatment is performed using the oil-based sealant It describes what can be done. However, the document does not describe or suggest a method of suppressing the elution of the fluorescent label into the oil-based encapsulant without performing the fixing process.

  In particular, in the conventional sample post-treatment process described in Patent Document 3 and the like, the stained tissue section is immersed in "an aqueous ethanol solution" to be dehydrated, and then dipped in xylene to be transparent, It was immersed in the sealing solution and sealed. For example, in dehydration processing, the amount of ethanol in an aqueous ethanol solution is increased to 50%, 70%, 90%, almost 100%, etc. (Conversely, the proportion of water is 50%, 30%, The tissue sections are sequentially immersed in their aqueous ethanol solution, and water in the tissue is replaced with ethanol, and then immersed in xylene in the clearing step, It was substituted by xylene. It is considered that the treatment with such an "ethanol aqueous solution" contributes to the problem of releasing the phosphor-accumulated nanoparticles.

  In addition, Patent Document 3 and the like describe the entrapment treatment in the case where staining treatment is performed by immunostaining targeting a protein, but in the case where staining treatment is performed by FISH targeting a gene (nucleic acid) Encapsulation treatment is not described, and it solves the problem of liberation of the probe labeled with phosphor-accumulated nanoparticles and the problem of elution of water-based staining reagent often used together when staining with FISH. There is no mention about that.

  “After dehydration without using aqueous ethanol solution, solvent substitution is carried out, and it is not necessary to separately fix at this time. In the present invention, conventional dehydration using“ ethanol aqueous solution ”and subsequent organic solvents are carried out It is clearly distinguished from the prior art that requires clear processing and fixation processing.

That is, the present invention includes the inventions represented by the following embodiments.
[1]
Treatment to fluorescently label the target biological substance with phosphor-accumulated nanoparticles based on FISH method or immunostaining method (FISH / immunostaining treatment), treatment to stain the nucleus with aqueous staining reagent (nuclear staining treatment), stained tissue A process of replacing the aqueous solvent remaining on the section with an oil-based solvent (solvent replacement process) and a process of encapsulating the tissue section with the solvent substituted with an oil-based sealant (encapsulation process) are performed in this order. Method of mounting the stained slide by FISH method or immunostaining method.
[2]
Item 2. The method for encapsulating a stained slide according to item 1, wherein the phosphor-aggregated nanoparticles are hydrophilic.
[3]
Item 3. The method for encapsulating a stained slide according to item 1 or 2, wherein the fluorescent substance-incorporated nanoparticle is an accumulation of a fluorescent dye consisting of a low molecular weight organic compound.
[4]
Item 5. The method for enclosing a dyed slide according to any one of Items 1 to 3, wherein the phosphor-accumulated nanoparticles are based on melamine resin.
[5]
Item 5. The method for encapsulating a dyed slide according to any one of Items 1 to 4, wherein the oil-based solvent is xylene.
[6]
At the time of the solvent substitution treatment, treatment (dehydration treatment) is carried out to dehydrate the tissue section with a dehydration treatment liquid comprising a mixed solvent containing an organic solvent having affinity for water having dehydration action and the oil solvent. The enclosure method of the dyeing | staining slide as described in any one of claim | item 1 -5.
[7]
Item 7. The method for sealing a dyed slide according to item 6, wherein the dewatering solution substantially does not contain water.
[8]
Item 8. The method for sealing a dyed slide according to item 7, wherein the dehydration processing solution is a mixed solvent of dehydrated ethanol and xylene.
[9]
A washing treatment solution comprising a mixed solvent containing a solvent in which the aqueous staining reagent used in the nuclear staining treatment does not elute, and an organic solvent blended in the dehydration treatment solution used in the dehydration treatment before the dehydration treatment The sealing method of the dyeing slide as described in any one of claim | item 6-8 which performs the process (washing process) to wash | clean.
[10]
Item 10. The method for encapsulating a staining slide according to item 9, wherein the aqueous staining reagent used in the nuclear staining step is DAPI, and the solvent in which the aqueous staining reagent is not eluted is PBS.

  According to the encapsulation method of the present invention, after staining by the FISH method or immunostaining method using the phosphor-accumulated nanoparticles or nuclear staining using a water-based staining reagent such as DAPI, it is stained by an oil-based mounting agent Even if the slide is sealed, it is possible to suppress the release of the phosphor-accumulated nanoparticles and the elution of the nuclear staining reagent. Thereby, it becomes possible to acquire a fluorescence image excellent in the recognition of the signal corresponding to the target gene or the target protein with less noise, and to quantify the expression level of the target gene or protein more accurately. , Leads to increase the reliability of pathological diagnosis. In particular, when the staining slide stored for a while after the staining treatment is observed to take a fluorescence image, the above-described problems are likely to occur, and therefore the effectiveness of the encapsulation method of the present invention is high.

FIG. 1 is a schematic view of a dyed slide represented in contrast to the suppression of the release of phosphor-accumulated nanoparticles, which is the first effect of the present invention. [A] and [B] are partial enlarged views of a region surrounded by a dotted circle in the cross-sectional view of the staining slide 1 in the upper part of FIG. [A] Dyeing slide 1 of the prior art sealed using water-based mounting medium 5a. It shows how the phosphor-accumulated nanoparticles 4 are released from the tissue section 3. [B] Dyeing slide 1 of the present invention sealed using oil-based mounting medium 5b. It shows a state in which the release of the phosphor-accumulated nanoparticles 4 from the tissue section 3 is suppressed. FIG. 2 is a schematic view of a staining slide represented in contrast to the elution of a water-based staining reagent, which is the second effect of the present invention. [A] and [B] are partial enlarged views of a region surrounded by a dotted circle in the cross-sectional view of the staining slide 1 at the top of FIG. [A] Dyeing slide 1 when washing processing is performed using conventional washing processing solution (for example, aqueous ethanol solution) 8a prior to dehydration processing. It shows a state where the aqueous staining reagent bound to the stained area 7b of the nucleus (chromosome) is eluted in the surrounding area 7a. [B] Staining slide 1 when the washing treatment is performed using a predetermined washing treatment solution (for example, a PBS / ethanol mixed solvent) prior to the dehydration treatment. It shows a state in which the elution of the aqueous staining reagent bound to the stained region 7b of the nucleus (chromosome) is suppressed. FIG. 3 is a flow chart showing an example of the embodiment in the case of performing staining based on the FISH method of the first embodiment of the method for enclosing a staining slide of the present invention. FIG. 4 is a flow chart showing an example of the embodiment in the case of performing staining based on the immunostaining method, of the second embodiment of the method for enclosing a staining slide of the present invention. FIG. 5 shows a fluorescence image obtained in each of Example 1 and Comparative Example 1 (a synthetic image obtained by superimposing a fluorescence image of FISH staining using fluorescent dye-integrated melamine particles and a fluorescence image of nuclear staining using DAPI) ). [A] Comparative Example 1: Case of sealing using water. In the circle indicated by the arrow, there is a bright spot of the fluorescent dye-aggregated melamine particle which has been released freely. [B] Example 1: Case of encapsulating using an oil-based encapsulant. FIG. 6 is a fluorescence image (a composite image similar to FIG. 5) acquired in each of Example 2 and Example 3. [A] Example 3: When washing with a PBS / ethanol mixed solvent was not performed (encapsulation using an oil-based mounting agent is performed). Although release of the fluorescent dye accumulated melamine particles is suppressed, extranuclear elution of DAPI is observed. [B] Example 2: In the case of washing with a PBS / ethanol mixed solvent (encapsulation using an oil-based mounting agent is also performed). While the release of fluorescent dye accumulated melamine particles is suppressed, the extranuclear elution of DAPI is suppressed.

  In the method for encapsulating a staining slide of the present invention, an embodiment in which the staining process is performed based on the FISH method (herein referred to as "first embodiment") and an embodiment in which the staining process is performed based on the immunostaining method (the present specification) Can be roughly classified into “second embodiment”.

In all of the first embodiment and the second embodiment, the entire process for preparing the preparation can be mainly classified into the "sample pretreatment process", the "staining process" and the "specimen post-treatment process".
The sample pretreatment step of one embodiment relating to the FISH method generally includes deparaffinization, pretreatment for FISH, enzyme treatment and the like.

  In the staining step of the first embodiment, a treatment to carry out staining based on FISH method (FISH treatment), that is, DNA denaturation treatment, hybridization treatment, post hybridization washing treatment, etc., and nuclear staining treatment (for example, by DAPI) included.

The sample post-treatment step of the first embodiment includes a solvent substitution treatment, an oil-based encapsulant encapsulating treatment, and if necessary, a washing treatment and a dehydration treatment to be performed before the solvent substitution treatment.
The sample pretreatment step of the two embodiments relating to the immunostaining method generally includes deparaffinization treatment, antigen activation treatment, washing treatment and the like.

  In the staining step of the second embodiment, a treatment to carry out staining based on immunostaining (immunostaining treatment), that is, a primary antibody depending on whether the target biological substance is directly labeled or indirectly labeled Treatment, secondary antibody treatment, staining treatment for morphological observation (including nuclear staining treatment), and the like are included.

  In the sample post-processing step of the second embodiment, as in the sample post-processing step of the first embodiment, a solvent replacement process, an oil-based encapsulant encapsulating process, and if necessary, a washing process performed before the solvent replacement process and Dehydration treatment is included.

  Hereinafter, the processes necessary to carry out the present invention will be further described. For matters not specifically described herein, for example, general items of processes and processes required to complete a preparation prepared on the basis of a FISH method or an immunostaining method, and a complete preparation. About the observation / photography process which had been done, the image processing / measurement process etc. which used the photographed image, it can be made appropriate according to the description items of patent documents 1-4 and other general technical matters. .

(Phosphor accumulated particles)
In the present invention, phosphor-aggregated nanoparticles are used as a substance for fluorescently labeling a target gene or protein. The phosphor-integrated nanoparticles are nano-sized by integrating a plurality of phosphors such as fluorescent dyes and semiconductor nanoparticles (quantum dots) into a matrix material or attaching them to the surface ((2) It is a particulate phosphor having a diameter of 1 μm or less. The use of such phosphor-accumulating nanoparticles in FISH or immunostaining is aimed at compared to using the fluorophore alone (fluorescent dye as one molecule or semiconducting nanoparticle as one particle) It is possible to enhance the intensity of the fluorescence emitted by each fluorescent label labeled with a biomolecule, to improve the discrimination between noise such as cell autofluorescence and other dyes, and to suppress fading due to irradiation of excitation light. It is preferable because it can be done.

  As a fluorescent substance which comprises fluorescent substance accumulation | occurence | production nanoparticle, the substance which can emit fluorescence by one molecule or one particle, such as a fluorescent dye and a semiconductor nanoparticle, can be used. A phosphor that emits fluorescence (color) of a desired wavelength in a stained image to be photographed may be selected. In addition, in the case where there are a plurality of biomolecules to be targeted for fluorescent labeling, a plurality of types of fluorescent substances that emit fluorescence of different wavelengths corresponding to each may be used in combination.

  As a fluorescent dye, for example, a fluorescein dye, a rhodamine dye, Alexa Fluor (registered trademark, manufactured by Invitrogen), a BODIPY (registered trademark, manufactured by Invitrogen), a cascade (registered trademark, Invitrogen) dye Dyes consisting of low-molecular-weight organic compounds (not high-molecular-weight organic compounds such as polymers), coumarin-based dyes, NBD (registered trademark) -based dyes, pyrene-based dyes, cyanine-based dyes, perylene-based dyes, and oxazine-based dyes Can be mentioned. Among them, sulforhodamine 101 and its hydrochloride, rhodamine dyes such as Texas Red (registered trademark), and perylene dyes such as perylene diimide are preferable because of relatively high light resistance.

  The semiconductor nanoparticles include, for example, particles consisting of a II-VI compound, a III-V compound, or a group IV element, or a core / shell type having these particles as a core and surrounded by a compound serving as a shell. (For example, CdSe / ZnS particles in which the core is CdSe and the shell is ZnS).

  As a base material which comprises a fluorescent substance accumulation | occurence | production nanoparticle, the substance which can integrate a fluorescent substance by physical or chemical binding force, such as resin and a silica, 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, AS resin (acrylonitrile-styrene copolymer) And various homopolymers and copolymers prepared using one or two or more monomers such as ASA resin (acrylonitrile-styrene-methyl acrylate copolymer). Among them, melamine resin and styrene resin are preferable because they can easily produce nanoparticles in which fluorescent substances such as fluorescent dyes are accumulated, and nanoparticles having high emission intensity can be obtained.

  As an example of the embodiment of the fluorescent substance accumulation nanoparticle, fluorescent dye accumulation resin particle produced using fluorescent dye as fluorescent substance and using resin as base; using fluorescent dye as fluorescent substance and using silica as base Fluorescent dye-integrated silica particles to be produced; semiconductor nanoparticle-integrated resin particles produced using a semiconductor nanoparticle as a fluorescent substance and a resin as a base; semiconductor nanoparticle-produced resin particles produced as a fluorescent substance; Semiconductor nanoparticle-integrated silica particles and the like.

  For example, a fluorescent dye-accumulating resin particle produced using a fluorescent dye such as perylene diimide, sulforhodamine 101 or its hydrochloride salt (Texas red) as a fluorescent substance and a resin such as a melamine resin or a styrene resin as a matrix It is preferable as a fluorescent substance accumulation | aggregation nanoparticle in this invention from having excellent performance etc.

  Furthermore, from the viewpoint of suppressing nonspecific adsorption due to hydrophobic bonding, etc., it is preferable that the phosphor-accumulated nanoparticles be hydrophilic. For example, a phosphor-accumulated nanoparticle is prepared using a hydrophilic substance as a matrix, such as melamine resin, or a phosphor-accumulated nanoparticle (a matrix is itself hydrophilic, such as melamine resin, but it is a styrene resin) Hydrophilic fluorescent substance-accumulated nanoparticles can be obtained by modifying the surface of (which may be hydrophobic) with a hydrophilic compound.

  The hydrophilic compound used for the surface modification of the phosphor-accumulated nanoparticles is not particularly limited. For example, linear hydrophilic polymers such as polyethylene glycol (PEG) and polypropylene glycol (PPG) have the number of repeating units. It is preferable from the viewpoint that the adjustment of the molecular length is easy, and that it is easy to prepare derivatives obtained by linking various functional groups etc. at the end or to obtain as a product.

  As an example, the method of hydrophilizing the fluorescent substance accumulation | storage nanoparticle which makes a styrene resin-type copolymer a base is as follows. By a copolymerization reaction using a compound having a predetermined functional group as a part of the monomer, and if necessary after the copolymerization reaction, the reaction of converting the functional group to a predetermined functional group to convert it into another functional group Further, a styrene resin copolymer having a functional group such as an amino group, a carboxyl group or a hydroxyl group on the particle surface can be prepared. The produced styrene resin copolymer (for example, one having an amino group) and a hydrophilic compound having a reactive group capable of binding to the functional group at one end (for example, N-hydroxysuccinimide-linked PEG) By reacting, the surface of the styrene resin copolymer can be modified with the hydrophilic compound, and hydrophilic phosphor-accumulated styrene resin particles can be obtained.

  The average particle size of the phosphor-aggregated nanoparticles is usually 10 to 500 nm, preferably 50 to 200 nm, and the variation coefficient of the particle size is usually 20% or less, preferably 5 to 15%. In addition, the particle size of the fluorescent substance accumulation | occurence | production nanoparticle image | photographs an electron micrograph with a scanning electron microscope (SEM), measures the cross-sectional area of resin particle for fluorescent labeling, and the area of the circle corresponded to the measured value. It can measure as a diameter (area circle equivalent diameter) when. The average particle size and coefficient of variation for the population of phosphor-loaded nanoparticles were determined as described above for each of a sufficient number (eg, 1000) of phosphor-loaded nanoparticles, the average particle size being Calculated as an arithmetic mean, the coefficient of variation is calculated according to the formula: 100 × standard deviation of particle size / average particle size.

  The phosphor-integrated nanoparticles as described above are known, and the phosphors used for the production and details of the matrix, the production method and the like, and specific examples of the embodiment are described in, for example, International Publication WO2013 / 035703 pamphlet, International Publication WO2013 Reference can be made to the publication No. 147081 pamphlet, the international publication WO 2014/136776 pamphlet and the like.

<FISH processing>
The FISH process performed in the first embodiment of the present invention is a process of labeling a target gene with phosphor-accumulated nanoparticles based on the FISH method in the staining step.

There are various methods in the FISH method, and it is not particularly limited as long as tissue sections can be stained so that the target gene can be fluorescently labeled and used for pathological diagnosis etc. Things like:
A method of preparing a fluorescently labeled probe in which a fluorophore and a probe are linked, and directly labeling and staining a target gene with the fluorescently labeled probe (direct method);
Prepare a biotin-modified probe in which a probe and biotin are linked, and an avidin-modified fluorophore in which a fluorophore and avidin or streptavidin are linked, react the biotin-modified probe with the target gene, and then react the avidin-modified fluorophore. A method of indirectly fluorescently labeling and staining a target gene using an avidin-biotin reaction (indirect method).

  In the above-mentioned indirect method, instead of biotin and avidin, haptens (materials which do not have immunogenicity but show antigenicity and relatively low molecular weight capable of reacting with antibodies) and anti-hapten antibodies such as dicoxigenin and anti It is also possible to use a combination of dicoxigenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antigens, as well as other substances with similar specific reactivity.

  The FISH method may be performed according to standard procedures and processing conditions for each of the various methods as described above. Generally, the specimen slide on which the tissue section is placed may be immersed in one 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 fluorescently labeled probe, a biotin-modified probe, an avidin-modified fluorescent substance, etc. are dissolved, and a blocking agent such as BSA is added as needed, Therefore, it is possible to produce and to obtain as a commercial item. For example, by replacing dTTP of a DNA clone of a target gene with biotin-modified dUTP by the nick translation method, a biotin-modified probe can be prepared in which a plurality of biotins are introduced into a DNA probe.

  The target gene targeted for FISH is not particularly limited, but typically, a protein targeted for pathological diagnosis based on FISH, such as HER2, TOP2A, HER3, EGFR, P53, MET, etc. Choose from various cancer- and tumor-related genes (so-called biomarker genes), as well as cancer-related genes such as cancer growth factors, transcription regulators, growth regulators, and regulators be able to.

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

<Nuclear staining process>
The nuclear staining process mainly performed in the first 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 the staining step. By performing a nuclear staining process, it is possible to count the number of cells and, at the same time, to count the number of bright spots of the fluorescent substance-integrated nanoparticles in which the target gene in the nucleus (on chromosome) is fluorescently labeled.

  Representative aqueous stains for nuclei include, but are not limited to, DAPI (4,6-diamidino-2-phenylindole dihydrochloride), which is 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.

<Immuno staining process>
The immunostaining process performed in the second embodiment of the present invention is a process of labeling a target protein with fluorophore-accumulated nanoparticles based on the immunostaining method in the staining step.

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

  In the above avidin-biotin combined primary antibody method or avidin-biotin combined secondary antibody method, instead of biotin and avidin, a hapten (which has no immunogenicity but shows antigenicity and can be reacted with an antibody Molecular weight substances) and anti-hapten antibodies, eg combinations of dicoxigenin and anti-dicoxigenin antibodies, FITC (fluorescein isothiocyanate) and anti-FITC antigens, as well as other substances with similar specific reactivity it can.

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

  The target protein to be targeted for immunostaining is not particularly limited, but typically, proteins targeted for pathological diagnosis based on tissue immunostaining, such as HER2, TOP2A, HER3, EGFR, P53, For proteins such as MET, proteins derived from various other cancer / tumor related genes (so-called biomarker genes), cancer growth factors, transcription regulators, growth regulators, receptors for transcription regulators, etc. It can be selected from related proteins.

  Monoclonal and polyclonal antibodies (primary antibodies) against the target protein, and antibodies (secondary antibodies) against those antibodies can be prepared according to known methods, and can also be obtained as commercial products.

<Solvent substitution treatment>
The solvent replacement process performed in both the first embodiment and the second embodiment of the present invention is a process for replacing the aqueous solvent remaining on the stained tissue section with the oil solvent in the sample post-treatment step. . For example, when the stained tissue section is immersed in PBS to wash away the attached excess staining solution, etc., the pulled up tissue section is covered with PBS, but the water of such PBS is removed and the oil is removed. It is a treatment that substitutes with a system solvent so that the tissue section is covered.

  As the oil-based solvent used in the solvent substitution treatment, it is possible to use the same solvent as the enclosed solvent contained in the oil-based encapsulant described later, that is, the organic solvent immiscible with water. Usually, the same compound as the encapsulating solvent contained in the oil-based encapsulating agent is used as the oil-based solvent in the solvent substitution step, but it is also possible to use a different compound if it does not inhibit the effects of the present invention.

  As such an oil-based solvent, one or more species satisfying the above conditions are selected from aromatic hydrocarbons, compounds (ketones) containing unsaturated hydrocarbon groups, esters, ethers and alcohols. Can be used. As an example, xylene, which is widely used as a solvent for oil-based encapsulant, is preferable as an oil-based solvent used in the solvent substitution treatment.

  The method for replacing the aqueous solvent with the oil-based solvent is not particularly limited. For example, after a tissue section to which an aqueous solvent such as water or buffer solution is attached is dried by air drying or the like to remove the aqueous solvent, immersing in an oil-based solvent contained in a container is one implementation of the solvent substitution treatment. It is a form. In addition, after a tissue section to which an aqueous solvent such as water or buffer solution is attached is preferably subjected to a washing treatment (details will be described later) and then a dehydration treatment using a specific dehydration treatment liquid (details to be described later) Immersion in an oil-based solvent contained in a container is also an embodiment of the solvent replacement process. The immersion in an oil-based solvent may be carried out usually at room temperature, and the immersion time is usually 2 to 10 seconds, preferably 2 to 5 seconds.

<Encapsulation process>
In the encapsulation process performed in both the first embodiment and the second embodiment of the present invention, the tissue section in which the aqueous solvent is replaced with the oil solvent by the solvent replacement process in the specimen post-treatment step is It is processing to use and enclose. Common mounting agents include water-based mounting agents and oil-based mounting agents (non-water-based mounting agents), but in the present invention, the latter oil-based mounting agents are used. Oil-based encapsulant has a small difference in refractive index from the sample, so it can be made transparent, it has good color and color of morphological staining, it is easy to prepare permanent samples, and it can be made more dewatered environment, so it can be used during sample preparation It is easy to prevent the mixing of water, and it is possible to make the drying at the time of preparation of the sample uniform.

  Although the method and conditions for the encapsulation treatment using the oil-based encapsulant are not particularly limited, generally, the oil-based encapsulant is dropped onto the tissue section on the specimen slide, and the cover glass is placed It may be dried. The sealed sample slide obtained by the above processing becomes a preparation to be used for pathological diagnosis and the like, and the preparation process is completed.

(Oil-based encapsulant)
The oil-based encapsulant is a mixture of a solvent (encapsulation solvent) containing an organic solvent immiscible with water and a resin soluble in the solvent. The oil-based encapsulant may be commercially available or may be prepared in-house. As a commercially available oil-based encapsulant, for example, DPX (manufactured by Sigma-Aldrich, main component: about 21.8% of polystyrene polymer, about 69.7% of xylene polymer), ENTERANNEW (registered trademark, manufactured by Merck) main component: Acrylic resin, xylene about 60%), Paramount N (registered trademark, manufactured by Pharma Co., main component: acrylic resin, aliphatic hydrocarbon) can be mentioned. Among commercially available oil-based encapsulants, there are those which can use the product as it is, and those which are used after diluting the product (stock solution) with a predetermined solvent. In addition, an oil based encapsulant can be prepared in house by dissolving a natural resin such as Canadian balsam or the like, or a synthetic resin such as polystyrene or polymethyl methacrylate in the encapsulating solvent.

  The encapsulating solvent contained in the oil-based encapsulating agent is a solvent containing an organic solvent immiscible with water, and the solvent contained in the commercially available oil-based encapsulating agent, a predetermined solvent for diluting the commercially available oil-based encapsulating agent And solvents used to prepare the oil-based mounting agent in-house. Here, "freely mixed with water" means that the solubility in water is 15% by volume or less. In the present invention, the above-mentioned “oil-based solvent” is used as the “oil-based solvent” used in the solvent substitution process performed prior to the oil-based encapsulant-encapsulating process and the “oil-based solvent” added to the dehydrating solution used in the dehydrating process. Similar materials to the "encapsulating solvent for the system encapsulant" can be used.

  As an organic solvent which is not freely miscible with water, aromatic hydrocarbon, compound (ketone) containing unsaturated hydrocarbon group, ester, ether, alcohol, among which one satisfying the above-mentioned solubility condition is selected. The above can be selected and used.

  Specific examples of the aromatic hydrocarbon include benzene, toluene and xylene. Examples of unsaturated hydrocarbons include limonene and pinene. As specific examples of the ketone, for example, hex opening hexanone, methyl ethyl ketone and the like can be mentioned. As an example of ester, butyl acetate etc. can be mentioned. Specific examples of the ether include anisole, 1,4-di (2-hydrioxyethoxy) benzene, ethylene glycol monophenyl ether and the like. Specific examples of the alcohol include butanol, pentanol, hexanol and the like. Among these compounds, xylene, toluene, limonene, etc. are easily available, have a refractive index of about 1.5 that is close to that of a tissue section, and a drying speed suitable for operation because they are dried in several tens of minutes. It is preferable from the point of having etc.

  Since the resin contained in the oil-based encapsulant is a substance which remains as a solid after evaporation of the encapsulating solvent, it is suitable to be one which does not affect observation, that is, one having a refractive index close to glass and high transparency. A resin that is colored or that itself fluoresces when it is irradiated with excitation light on a fluorescent label (fluorescent substance-accumulating nanoparticles) adversely affects fluorescence microscopy and should not be included. . As preferable resin which satisfy | fills such conditions, for example, natural resin like a Canadian balsam etc., and synthetic resin like a polystyrene, polymethyl methacrylate, etc. are mentioned.

  An anti-fading agent can be blended with the oil-based encapsulant, if necessary. As the anti-fading agent for that purpose, for example, among phenol-based, amine-based, phosphorus-based, sulfur-based and unsaturated hydrocarbon-based anti-fading agents, it is possible to use an oil-based encapsulant, that is, a solvent immiscible with water. One or two or more types that do not have a problem in solubility in the enclosed solvent can be selected and used. Such an anti-fading agent also has no absorption in the range of 450 to 600 nm overlapping with the excitation light wavelength (peak wavelength) of the fluorescent label (phosphor-accumulated nanoparticles) so as not to adversely affect fluorescence microscopy. It is preferable that there is no light emission at 500 to 700 nm overlapping with the emission wavelength of the fluorescent label.

<Dehydration treatment>
The dehydration treatment performed in both the first embodiment and the second embodiment of the present invention is carried out in the sample post-processing step, including the water contained in the stained tissue section, which is optionally performed in the solvent replacement treatment. It is a process for removing as much as possible. Such dehydration treatment has conventionally been generally performed using a mixture of dehydrated ethanol and water, but in the present invention, it contains a specific organic solvent and an oil-based solvent as described below. It is carried out using a dewatering solution comprising a mixed solvent.

(Dehydration solution)
The dehydration treatment liquid in the present invention is a mixed solvent containing an organic solvent having a high affinity for water having a dehydration action and an oil-based solvent. A dehydrating solution which satisfies such conditions can be prepared by mixing an appropriate organic solvent and an oil-based solvent in an appropriate ratio as described below.

  It is preferable that the dewatering solution is not substantially compatible with the oil-based solvent, and that it is "substantially free" of "water" which may cause elution of the aqueous staining reagent used in the nuclear staining process. "Substantially free" means that it is acceptable to include a trace amount of water which is difficult to completely remove in the preparation of a dehydrating solution, such as water contained in a trace amount in dehydrated ethanol described later. Means

  As the oil-based solvent contained in the dehydrating treatment solution, the same solvent as the sealing solvent contained in the above-mentioned oil-based sealant, that is, the organic solvent immiscible with water can be used. However, this oil-based solvent does not have to be mixed freely with water itself (without having compatibility with water itself), but it freely mixes with an aqueous solvent freely mixed with water (other than water Compatibility with the aqueous solvent of Usually, the same compound as the oil-based solvent used in the solvent-containing solvent and the encapsulating solvent contained in the oil-based encapsulant is used as the oil-based solvent to be compounded in the dehydrating treatment liquid, It is also possible to use a compound different from them as an oil-based solvent which is compounded in the dehydration treatment liquid.

  As such an oil-based solvent, one or more selected from the group consisting of aromatic hydrocarbon, unsaturated hydrocarbon carbonyl-containing compound (ketone), ester, ether and alcohol, which satisfy the above conditions It can be used. As an example, xylene having compatibility with ethanol, which is a representative example of an aqueous solvent to be described next, is preferable as an oil-based solvent to be blended in the dehydration treatment liquid.

  As the organic solvent contained in the dehydrating treatment solution, a compound having a dehydrating action similar to a general dehydrating agent can be used. Such an organic solvent should have compatibility with water, be freely miscible, be compatible with the aforementioned oil-based solvent, and be freely miscible. As an example, ethanol having a dehydrating action and having compatibility with xylene, which is a typical example of the oil-based solvent described above, is preferable as an organic solvent to be blended in the dehydration treatment liquid.

  However, the "ethanol" used in the preparation of the dehydrating solution of the present invention is substantially free of water, that is, not an "ethanol aqueous solution", and dewatered ethanol (ethanol concentration 99.5% or higher) or high purity according thereto Ethanol, which does not contain water in the dehydrating solution prepared using it.

  The dehydration treatment may be performed multiple times using a dehydration treatment liquid in which the ratio of the organic solvent to the oil-based solvent to be mixed is changed in several steps. For example, two types of mixed solvents of ethanol: xylene = 50: 50 and 20:80 may be prepared on a volume basis, and dehydration processing may be sequentially performed using the respective mixed solvents. Subsequent to the dehydration treatment, a solvent substitution treatment is carried out, in which treatment with an oil solvent containing only the same compound as the oil solvent contained in the dehydration treatment liquid (for example, only xylene) is carried out. It is appropriate to use the liquid in the order of the liquid having a high proportion of the organic solvent and a low proportion of the oil-based solvent, to the one having a low proportion of the organic solvent and a high proportion of the oil-based solvent.

  Although the method for dehydration treatment using the dehydration treatment solution is not particularly limited, in general, the tissue section may be immersed in the dehydration treatment solution contained in a container. The immersion in the dehydrating solution may be carried out usually at room temperature, and the immersion time is usually 2 to 10 seconds, preferably 2 to 5 seconds.

<Cleaning process>
The washing treatment mainly performed in the first embodiment of the present invention is a treatment performed as needed before dehydration treatment in the sample post-treatment step, and a washing treatment liquid comprising a specific aqueous mixed solvent is used. And washing the stained tissue section. When a nuclear staining process is performed using an aqueous staining reagent such as DAPI, it is preferable to perform such a washing process, and the excess aqueous staining reagent is washed away beforehand, and the dehydration process used in the dehydration process is carried out. It does not elute in oil-based solvents used in solution or solvent replacement treatment.

  In the preparation process of the preparation, the sample slide may be washed after processing the sample slide with various solutions, but the washing process according to the present invention may be performed after the nuclear staining process using the aqueous staining reagent. , Refers to what is done before dehydration processing.

(Cleaning solution)
The cleaning solution used for the cleaning process of the present invention can be prepared by mixing the two types of solvents described below in appropriate proportions.

  The first solvent for the cleaning solution is a solvent in which the aqueous staining reagent used in the nuclear staining process does not elute. For example, DAPI, which is a typical aqueous staining reagent, uses pure water as a solvent for the reagent, but once stained (intercalated) on the target DNA, it is PBS (phosphate buffered saline). Hardly dissolves. Therefore, when performing nuclear staining using DAPI, since unreacted DAPI can be washed away by adding PBS, PBS can be used as the first solvent.

As the second solvent for the washing treatment liquid, the same solvent as the organic solvent contained in the above-described dehydration treatment liquid, for example, dehydrated ethanol can be used. Isopropanol, acetone or the like can be used instead of dehydrated ethanol.
Therefore, for example, a mixed solution of PBS (first solvent) and dehydrated ethanol (second solvent) can be used as the washing solution.

  The washing treatment may be performed a plurality of times using a dehydration treatment liquid in which the ratio of the aqueous solvents to be mixed is changed in several steps. For example, when PBS is used as the first solvent and dehydrated ethanol is used as the second solvent, a mixed solution of two types of PBS: dehydrated ethanol = 50: 50 and 20:80 is prepared, and The mixed solution may be sequentially used for washing treatment. Subsequent to the washing treatment, the dehydration treatment is carried out, where the treatment using the dehydration treatment solution containing the second solvent contained in the washing treatment solution is carried out, so the washing treatment solution has a high ratio of the first solvent and the first treatment It is appropriate to use in order of the ratio of the two solvents being low to the ratio of the first solvent being low and the ratio of the second solvent being high.

  Although the method for the washing treatment using the washing treatment solution is not particularly limited, in general, the tissue section may be immersed in the washing treatment solution contained in the container. The immersion in the cleaning solution may be carried out usually at room temperature, and the immersion time is usually 2 to 10 seconds, preferably 2 to 5 seconds.

<I. Preparation of fluorescently labeled probe for HER2 gene>
(1) Preparation of Biotin-Modified BAC Probe CellBiochemBiophys. A biotin-modified BAC probe was prepared by the nick translation method according to the method described in 2006; 45 (1) 59. That is, a kit for nick translation (product name "GSP-nick translation kit" K- 015, manufactured by GSP) with respect to 1 μg (5 μL) of HER2-DNA clone (about 150 kbp) purchased from GSP is used as a protocol The dTTP of the HER2-DNA clone (nucleic acid molecule) was replaced with biotin-modified dUTP according to. The specific preparation procedure is as follows.

First, the following reagents were mixed in a centrifuge tube.
10 × Nick Buffer (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 No. 1093070, manufactured by Roche, 50 nmol / 50 μL): 0.2 μL
-Pure water (Nuclease free water)-3 μL
・ The above-mentioned HER2-DNA clone approximately 150 kbp) 1 μg of aqueous solution ... 5 μl
· DNA lylymerase I (Tris-HCl [pH 7.5], EDTA, DTT
, Glycerol) ... 1 μL
・ DNase I ... 5 μL

  Next, the above 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 by a microspin column for purification of nucleic acid ("MicroSpin S-200 HR Column" manufactured by GE Healthcare, product number "# 27-5120-01").

  About 5.56 microliters of 3M sodium acetate solution (pH 5.2) and 150 microliters of 100% ethanol were added with respect to this solution, and it left still at -20 degreeC for 1 hour or more. The precipitate was formed by centrifugation at 16000 rpm at 4 ° C. for 10 minutes. Furthermore, 500 μL of 70% ethanol was added, and the supernatant was removed by centrifugation at 16000 rpm for 1 minute at 4 ° C. The precipitate was completely dissolved by adding 5-10 μl of distilled water to obtain a solution of biotin-modified BAC probe with a final concentration of 1 μg / 250 μl.

(2) Preparation of Streptavidin-Modified Fluorescent Dye Accumulated Melamine Resin Particles
20.3 mg of “Sulfo Rhodamine 101” (manufactured by Sigma Aldrich) was added to and dissolved in 22 mL of water. Thereafter, 2 mL of a 5% aqueous solution of an emulsifier for emulsion polymerization "Emulgen" (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation) was added to this solution. The solution was heated to 70 ° C. while being stirred on a hot stirrer, and then 0.81 g of a melamine resin raw material “Nicalac MX-035” (manufactured by Nippon Carbide Industries Co., Ltd.) was added to the solution. Furthermore, 1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Ltd.) as a surfactant was added to this solution, and the mixture was heated and stirred at 70 ° C. for 50 minutes. Thereafter, the temperature was raised to 90 ° C., and heating and stirring were performed for 20 minutes.

  Washing with pure water was performed to remove impurities such as excess resin raw materials and fluorescent dyes from the obtained dispersion liquid of fluorescent dye accumulated melamine resin particles. Specifically, the mixture is centrifuged at 20000 G for 15 minutes in a centrifuge "Micro Cooled Centrifuge 3740" (manufactured by Kubota Corporation), and after removing the supernatant, ultrapure water is added and ultrasonicated for redispersion. did. Washing by centrifugation, supernatant removal and redispersion in ultrapure water was repeated 5 times.

  0.1 mg of the above-mentioned fluorescent dye accumulated melamine resin particles is dispersed in 1.5 mL of ethanol, 2 μL of aminopropyltrimethoxysilane (LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) is added, and reacted for 8 hours. A surface amination treatment was performed to convert hydroxyl groups present on the surface into amino groups.

The concentration of the fluorescent dye-integrated melamine resin particles was adjusted to 3 nM using phosphate buffer saline (PBS) containing 2 mM ethylenediaminetetraacetic acid (EDTA). SM (PEG) ₁₂ (Succinimidyl-[(N-maleimidopropionamide) -dodecaethylene glycol] ester, thermo-scientific agent so that the final concentration is 10 mM with respect to the dispersion of the concentration-adjusted fluorescent dye accumulation melamine resin particles The mixture is mixed at a temperature of 20 ° C. for 1 hour to obtain a mixed solution containing fluorescent dye-aggregated melamine resin particles whose particle surface is modified with a maleimide group.

  The mixture was centrifuged at 10000 G for 20 minutes, and after removing the supernatant, PBS containing 2 mM EDTA was added to disperse the precipitate, and centrifugation was performed again. After performing the above washing three times according to the same procedure, the maleimide group-modified fluorescent dye-aggregated melamine resin particles were recovered.

  On the other hand, after performing the addition processing of a thiol group to streptavidin using streptavidin (made by Wako Pure Chemical Industries Ltd.) and N-succimidyl-S-acetylthioacetic acid (SATA), gel filtration is carried out, and fluorescent substance Streptavidin capable of binding to the accumulated nanoparticles was prepared separately.

  The above-mentioned fluorochrome-incorporated melamine resin particles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react at room temperature for 1 hour to couple both (maleimido group and thiol group). Thereafter, 10 mM mercaptoethanol was added to stop the reaction. The resulting solution is concentrated with a centrifugal filter of φ = 0.65 μm, and then unreacted streptavidin and the like are removed using a gel filtration column for purification to obtain fluorescent dye-aggregated melamine resin particles which have been subjected to Streptavidin modification. The

(3) Preparation of a fluorescently-labeled body in which fluorescent dye-integrated melamine resin particle BAC probe is linked: 25 μL of a biotin-labeled BAC probe (concentration 1 μg / 250 μL) prepared as described above, and streptavidin-labeled fluorescent dye The solution of accumulated melamine resin particles was mixed, reacted at room temperature for 30 minutes, and biotin and each avidin were combined to obtain a DNA probe for HER-2 detection.

(4) Storage of fluorescently labeled DNA probe Hybridization buffer (25% deionized formamide, 2 × SSC, 200 ng / μL salmon sperm DNA, 5 × Denhardt's solution, 50 mM) for the fluorescently labeled DNA probe obtained as described above Sodium phosphate, pH 7.0, 1 mM EDTA) to a final concentration of 1 to 5 ng / μL. The free ligand (substrate for free streptavidin, biotin and dATP) was removed by a S300 sized spin column (Amersham Biosciences, Piscataway, NJ). The fluorescently labeled DNA probe was stored frozen at -20.degree. C. until use.

<II. Preparation of stained slide based on FISH method>
The copy number of HER2 gene was measured by FISH. As described below, FISH is deparaffinized, pretreated for FISH, enzyme treated (so far "sample pretreatment step"), preparation of probes, DNA denaturation treatment, hybridization treatment, post hybridization washing treatment (here "Staining step"), nuclear staining treatment with DAPI, solvent substitution treatment and encapsulation treatment (so-called "sample post-treatment step") were carried out in this order. It is referred to as Examples 1 to 3 and Comparative Example 1 according to the embodiment of the solvent substitution treatment and the encapsulation treatment.

Example 1
(1) Specimen pretreatment step (1-1) Deparaffinized HER2-positive stained specimen specimen slide (Roche HER2 Dual ISH 3-in-1 control slide Product code: 109530) to the following (i) to (i) Deparaffinization was performed according to the procedure of iv). (I) The resin is immersed in deparaffinizing agent "Hemo-De" (Pharma Co., Ltd., main component: d-limonene, antioxidant) for 10 minutes at normal temperature. (Ii) Immerse the specimen slide in fresh "Hemo-De" for 10 minutes at normal temperature. Repeat the same operation three times. (Iii) Immerse the specimen slide in 100% ethanol at normal temperature for 5 minutes, wash twice, and dehydrate. (Iv) Dry the specimen slide on air or on a slide warmer at 45 to 50 ° C.

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

(1-3) Enzyme treatment The sample slide subjected to the protein removal treatment 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 wash buffer. (Ii) Immerse the specimen slide in a protease solution heated to 37 ° C. for 10 to 60 minutes. This immersion treatment is carried out with 25 mg protease (2500-3000 Units / mg) [Pepsin] / 50 mL of 1 M NaCl [pH 2.0] for 60 minutes) to degrade proteins of cell membranes and nuclear membranes, particularly collagen. It was processed. (Iii) Immerse the specimen slide in wash buffer for 5 minutes. Repeat this operation twice. (Iv) The sample slide was air dried.

(2) Staining step (2-1) Preparation of probe The fluorescence-labeled DNA probe solution stored frozen is returned to room temperature, and the viscosity of the solution is sufficient to the extent that pipetting operation is possible for accurate volume collection. The solution was mixed using a vortex mixer.

(2-2) DNA denaturation treatment In order to denature DNA on a specimen slide, DNA denaturation treatment was performed on the specimen slide subjected to the fixation treatment according to the following procedures (i) to (viii). (I) A wet box (airtight container with its side sealed with a paper towel) covered with a paper towel moistened with water prior to the preparation of the specimen slide is placed in a 37 ° C incubator and reserved Heat up. (Ii) Confirm 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 normal temperature. Place the denaturing solution in a Coplin jar and warm in the warm bath until the denaturing solution is at 72 ° C. ± 1 ° C. (place in the 72 ± 1 ° C. warm bath for at least 30 minutes). (Iii) Mark the area on the back of the sample slide with a diamond pen or the like so that the hybridization area can be identified. (Iv) Immerse the specimen slide in a Coplin jar containing denaturing solution at 72 ± 1 ° C. to denature the DNA of the specimen slide. (V) Remove the specimen slide from the denaturing solution using tweezers and immediately place in 70% ethanol at ambient temperature. Shake the slide to remove formamide. Soak the specimen slide for 1 minute. (Vi) Remove the specimen slide from 70% ethanol, place in 85% ethanol and shake the slide to remove formamide. Soak the sample for 1 minute. Repeat the same operation twice with 100% ethanol. (Vii) Place the lower end of the specimen slide glass on 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) Hybridization treatment The sample slide subjected to the denaturation treatment was subjected to a hybridization treatment of a fluorescently labeled DNA probe in the following procedures (i) to (iii). (I) Add 10 μL of fluorescently labeled DNA probe solution onto the tissue section of the sample slide, immediately cover it with a 22 mm × 22 mm cover glass, and make the fluorescently labeled DNA probe solution uniform while preventing air bubbles from entering. spread. (Ii) Seal the cover glass with a paper bond. (Iii) Place the specimen slide in a pre-warmed wet box, cover, and perform hybridization for 14-18 hours in a 37 ° C. incubator.

(2-4) Post-hybridization washing treatment Post-hybridization washing treatment was performed on the sample slide subjected to the hybridization treatment according to the following procedures (i) to (vi). (I) Post-hybridization wash buffer (2 × SSC / 0.3% NP-40) is placed in the copier. Place in a warm bath at 72 ° C ± 1 ° C for at least 30 minutes and preheat in the warm bath until the post hybridization wash buffer is at 72 ° C ± 1 ° C (place in a warm bath at 72 ° C ± 1 ° C for at least 30 minutes ). (Ii) Prepare another Coplin jar containing post-hybridization wash buffer and maintain it at ambient temperature. (Iii) Remove the paper bond seal with tweezers. (Iv) Place the specimen slide in post-hybridization wash buffer. Wait for the coverslip to peel off in solution naturally. (V) Remove the specimen slide from the solution, remove excess solution, and soak in post-hybridization wash buffer warmed to 72 ± 1 ° C. for 2 minutes. (6) Take out the sample slide from the Coplin jar and air dry it in the dark.

(2-5) Nuclear Staining Treatment 10 μL of DAPI staining solution (2 μg / mL PBS, Molecular Probes, D1306) is added to the hybridization area of the sample slide subjected to post-hybridization washing, and the reaction is continued for 10 minutes at 25 ° C. It hold | maintained and performed the nuclear-staining process by DAPI.

(3A) Sample post-treatment step (3A-1) Solvent substitution treatment After air-drying the specimen slide subjected to the nuclear staining treatment for 60 minutes at normal temperature, the solvent substitution is carried out by immersing in xylene for 2 to 10 seconds at normal temperature. I did the processing.

(3A-2) Encapsulation Treatment Entranum New (Merck) was dropped onto the tissue of the sample slide subjected to the solvent displacement treatment. The cover glass was put, and the sealing process was performed by air-drying at normal temperature for 10 minutes. Thereafter, the sample slide subjected to the encapsulation process was protected from light and stored until signal measurement.

Comparative Example 1
The specimen pretreatment step (1) and the staining step (2) were performed in the same manner as in Example 1 described above, and then the specimen post-treatment step (3A) was changed to the following treatment.
The sample slide subjected to the nuclear staining treatment was covered with a cover glass as it was, and air-dried at room temperature for 10 minutes. Thereafter, the sample slide was shielded from light and stored until signal measurement.

Example 2
After the sample pretreatment step (1) and the staining step (2) are performed in the same manner as in Example 1 described above, the sample post-treatment step (3A) is changed to the following sample post-treatment step (3B) The

(3B) Sample post-treatment step (3B-1) Washing treatment PBS: two types of PBS / dehydrated ethanol mixed solvents (50% and 50: 20% by volume of dehydrated ethanol (volume%) respectively (first PBS / dehydrated ethanol mixed solvent respectively) And a second PBS-dehydrated ethanol mixed solvent. The specimen slide subjected to the nuclear staining treatment was immersed in the first PBS / dehydrated ethanol mixed solvent for 2 seconds at normal temperature, and subsequently also immersed in the second PBS / dehydrated ethanol mixed solvent for 2 seconds.

(3B-2) Dehydration Treatment Dewatered ethanol: two kinds of dehydrated ethanol-xylene mixed solvents of 50: 50 and 20: 80 (% by volume of xylene) (respectively mixed ethanol of dehydrated ethanol and xylene and mixed second dehydrated ethanol and xylene) Prepared as a solvent). The specimen slide subjected to the washing treatment was immersed in the first dehydrated ethanol-xylene mixed solvent for 2 seconds at normal temperature, and subsequently immersed in the second dehydrated ethanol-xylene mixed solvent for 2 seconds.

(3B-3) Solvent Substitution Treatment The solvent displacement treatment was performed by immersing the specimen slide subjected to the dehydration treatment in xylene at normal temperature for 2 to 10 seconds in xylene.

(3B-4) Encapsulation treatment After dropping enthalang new (Merck) at room temperature, the sample slide subjected to the solvent substitution treatment is covered with a cover glass and air-dried at ambient temperature for 10 minutes to perform the encapsulation treatment. The Thereafter, the sample slide subjected to the encapsulation process was protected from light and stored until signal measurement.

[Example 3]
After performing the specimen pretreatment step (1) and the staining step (2) in the same manner as in Example 2 (that is, Example 1) described above, the specimen post-treatment step (except that the washing treatment (3B-1) was not performed The sample post-processing step (3B) was performed in the same manner as 3B).

<III. Shooting and Analysis of Fluorescent Images>
Observation of the stained slide prepared in 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: x 100 oil immersion) and A fluorescent image was taken (1000 ×). Fluorescent dye (sulforhodamine 101) accumulated melamine resin particles are irradiated with excitation light having an excitation wavelength of 575 to 600 nm using an appropriate filter, and fluorescence having a fluorescence wavelength of 610 to 670 nm is observed, and its fluorescence image I got DAPI irradiated the excitation light which makes an excitation wavelength 340-380 nm using a suitable filter, observed the fluorescence which makes a fluorescence wavelength 435-485 nm, and acquired the fluorescence image.

  The fluorescence images of Examples 1 to 3 and Comparative Example 1 are shown in FIGS. 5 and 6. In Example 1 (FIG. 5 [B]), Example 2 (FIG. 6 [B]) and Example 3 (FIG. 6 [A]) in which the encapsulation process using the oil-based encapsulant is performed, The release of melamine particles is suppressed. Moreover, in Example 2 (FIG. 6 [B]) which wash | cleaned by the PBS / ethanol mixed solvent, elution of DAPI is also suppressed. FIG. 5 is an enlarged view of a part of the 1000 × visual field image.

1: Stained slide 2: Slide glass 3: Tissue section 4: Phosphor-accumulated nanoparticles 5a: Water-based mounting medium 5b: Oil-based mounting medium 6: Cover glass 7a: Surrounding area of nucleus (chromosome) 7b: Nucleus (chromosome) Dyeing area 8a: Conventional cleaning solution 8b: Cleaning solution of the present invention

Claims (9)

Treatment to fluorescently label the target biological substance with phosphor-accumulated nanoparticles based on FISH method or immunostaining method (FISH / immunostaining treatment), treatment to stain the nucleus with aqueous staining reagent (nuclear staining treatment), stained tissue Treatment of washing sections with a mixed solution of PBS (phosphate buffered saline) and ethanol, treatment of replacing aqueous solvent remaining on stained tissue sections with oil-based solvent (solvent substitution treatment), and solvent A process for encapsulating a stained slide by the FISH method or immunostaining method, characterized in that a process (enclosing process) for encapsulating a substituted tissue section with an oil-based sealant is performed in this order.   The method for encapsulating a dyed slide according to claim 1, wherein the phosphor-accumulated nanoparticles are hydrophilic.   The method for enclosing a dyed slide according to claim 1 or 2, wherein the phosphor-accumulated nanoparticles are ones in which fluorescent dyes consisting of low molecular weight organic compounds are accumulated.   The method for enclosing a dyed slide according to any one of claims 1 to 3, wherein the phosphor-aggregated nanoparticles are based on melamine resin.   The method for encapsulating a dyed slide according to any one of claims 1 to 4, wherein the oil-based solvent is xylene.   At the time of the solvent substitution treatment, treatment (dehydration treatment) is carried out to dehydrate the tissue section with a dehydration treatment liquid comprising a mixed solvent containing an organic solvent having affinity for water having dehydration action and the oil solvent. The sealing method of the dyeing slide as described in any one of Claims 1-5.   The method for enclosing a staining slide according to claim 6, wherein the dehydration processing solution contains substantially no water.   The method for enclosing a staining slide according to claim 7, wherein the dehydration processing solution is a mixed solvent of dehydrated ethanol and xylene. Ru aqueous staining reagent DAPI der used in the nuclear stain treatment method of encapsulation stained slides according to any one of 請 Motomeko 1-8.