JP6237194B2 - Dyeing method - Google Patents

Description

  The present invention relates to a method for staining a pathological section, and more particularly to a technique capable of realizing both immunostaining and morphological observation staining.

  In pathological diagnosis, tissue specimens obtained by organ excision or needle biopsy are sliced to a thickness of several microns to create tissue specimens, and they are widely observed using an optical microscope to obtain various findings. It has been broken. In many cases, a specimen is prepared by dehydrating and collecting a paraffin block to fix the collected tissue, then slicing it to a thickness of several μm, and removing the paraffin. Here, since the specimen hardly absorbs and scatters light and is almost colorless and transparent, it is general that the specimen is stained with a dye prior to observation.

Various dyeing techniques have been proposed.
Especially for tissue specimens, hematoxylin and eosin staining (HE staining) using two pigments of hematoxylin and eosin is used as a standard morphological observation stain for observing the morphology of the specimen (Non-patent Document 1, Patent Document). 1-2), cell nucleus, lime, cartilage tissue, bacteria and mucus are stained blue-blue to light blue by hematoxylin staining (H staining), and cytoplasm, stroma, various fibers, etc. by eosin staining (E staining) Red blood cells and keratinocytes are stained red to dark red. The pathologist makes a diagnosis based on morphological information such as changes in the size and shape of the cell nucleus and changes in the pattern of the tissue in the microscopic image of the stained tissue specimen, and staining information. ing.

On the other hand, in pathological diagnosis, immunological observation called immunostaining is performed, in which molecular target staining for confirming the expression of molecular information in a tissue specimen is performed to diagnose functional abnormalities such as abnormal expression of genes and proteins.
In immunostaining, for example, a fluorescent staining method (fluorescent labeling method) is used. The fluorescent staining method is a technique for measuring the amount of antigen by staining and observing an antigen of interest using an antibody modified with fluorescent particles such as a fluorescent dye.

In recent years, it has been attempted to perform both morphological observation staining and immunostaining by fluorescence staining (fluorescence immunostaining) on the same pathological section. For example, when detecting proteins expressed on membranes such as HER2 In addition, HE staining and fluorescent immunostaining are performed together.
In this case, since it is desired to perform both cell morphology observation and fluorescent particle observation, the fluorescent particles are excited at an excitation wavelength at which eosin emits light to some extent. At that time, fluorescent particles with low luminance (for example, Q-dot) are buried with fluorescence due to E staining, making it impossible to observe a bright spot. Therefore, fluorescent particles having luminance not buried in E staining are selected. It will be necessary. In the fluorescent particles currently used, since the luminance is proportional to the volume of the particles, particles having a somewhat large particle size are necessary at the time of HE staining, and in reality, particles having a particle size of about 150 nm are required. It is used.

Special table 2001-525580 gazette JP 2009-115599 A

Immunohistochemistry useful for diagnosis, Bunkodo

By the way, although it is not a big problem in a pathological section with a small number of antigen expression, a problem arises in a pathological section with a large number of antigen expression because the distance between antigens may be 150 nm or less.
For example, as shown in FIG. 5, a labeled body 10 in which fluorescent particles 12 and biotin 14 are directly bound is prepared, and a secondary antibody 40 obtained by modifying the antigen 20 with a primary antibody 30 and an avidin 42 using the labeled body 10. In the method of immunostaining the combination of the fluorescent particles 12, since the fluorescent particles 12 have a particle size of about 150 nm, there are fluorescent particles 12 that cannot bind to the antigen 20 due to steric hindrance between the fluorescent particles 12, The antigen 20 cannot be recognized.
In response to such a problem, if only the fluorescent particles 12 having a small particle diameter are used, the fluorescent particles 12 can be recognized even when the distance between the antigens 20 is narrow, and the number of bright spots increases under the observation condition of only H staining. However, since the fluorescent particle 12 having a small particle size has low luminance itself, when both fluorescent immunostaining and HE staining are performed, the fluorescence of the fluorescent particle 12 is weaker than that of eosin, and the bright spot cannot be confirmed. Quantification by staining is reduced.

  Therefore, a main object of the present invention is to provide a staining method capable of improving the quantitativeness of an antigen by fluorescent immunostaining even when both fluorescent immunostaining and morphological observation staining are performed.

In order to solve the above problems, according to the present invention,
In a staining method of a pathological section using two or more kinds of fluorescent particles having different average particle diameters, which are fluorescent particles encapsulating a fluorescent dye in a resin,
Immunostaining a specific antigen with the first fluorescent particles having a first average particle diameter and emitting a first fluorescent color with respect to the pathological section;
A second fluorescent color having an average particle size smaller than the first average particle size of the first fluorescent particles and different from the first fluorescent color is applied to the pathological section . Immunostaining the specific antigen using second fluorescent particles that emit ;
There is provided a staining method characterized by comprising:

  According to the present invention, even when both fluorescent immunostaining and morphological observation staining are performed, the antigen quantification by fluorescent immunostaining can be improved.

It is a figure which shows roughly the relationship between the resin amount of fluorescent particles, and an average particle diameter. It is a spectrum figure which shows roughly the excitation spectrum and fluorescence spectrum of eosin. It is a schematic diagram schematically showing the behavior of particles during immunostaining using large fluorescent particles and small fluorescent particles. It is a figure which shows the relationship between the total protein amount (antigen amount) and the number of bright spots concerning the Example of this invention. It is a figure for demonstrating the problem of a prior art.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

The staining method according to a preferred embodiment of the present invention basically includes (1) a step of immunostaining a pathological section using fluorescent particles having a large average particle diameter, and (2) fluorescence having a small average particle diameter of the pathological section. A step of immunostaining using particles; and (3) a step of observing and staining a pathological section using a staining reagent for morphological observation.
In particular, in the step of immunostaining pathological sections (1) and (2), fluorescent particles having different average particle diameters are used as labeling agents. The pathological section after fluorescent staining is irradiated with excitation light to cause these fluorescent particles to emit fluorescence, and a biological substance (antigen) is detected from the pathological section.

In the immunostaining step (1) and the immunostaining step (2), the processing of the immunostaining step (1) is performed first, and then the processing of the immunostaining step (2) is performed. To do. For observation of bright spots of fluorescent particles, the number of bright spots of fluorescent particles with a large average particle diameter is counted under HE staining conditions, and then the number of bright spots of fluorescent particles with a small average particle diameter is changed by switching the excitation wavelength. Count.
On the other hand, in the immunostaining step (1) and (2) and the morphological observation staining step (3), preferably, the processing of the immunostaining step (1) and (2) is performed first, and then In addition, the process of (3) morphological observation dyeing process is preferably executed, but the order (first and second) may be changed.

  Details of immunostaining, the characteristics and types of fluorescent particles used therein, and morphological observation staining are as follows.

[Immunostaining]
(I) Staining method In immunostaining, a fluorescent staining method is preferably used.
The fluorescent staining method is a method of staining an antigen with fluorescent particles.
At the time of staining, a labeled body in which fluorescent particles and a primary antibody are directly bound is prepared, a method for staining an antigen (primary antibody method), a labeled body in which fluorescent particles and a secondary antibody are directly bound are prepared, A method in which a primary antibody bound to an antigen is stained (secondary antibody method), a labeled body in which fluorescent particles and biotin are directly bound is prepared, and a primary antibody and a secondary antibody modified with avidin are bound to the antigen. A method of staining the bound one (biotin-avidin method, see FIG. 5) or the like can be used.
The primary antibody is not particularly limited, and varies depending on the subject (antigen) to be immunostained. For example, when performing immunostaining of HER2 antigen, an anti-HER2 antibody is used.
“Antigen” is preferably a membrane protein or cytoplasmic protein.
The secondary antibody is not particularly limited and varies depending on the primary antibody. For example, anti-mouse, rabbit, cow, goat, sheep, dog, chicken.
Any existing method may be used for binding the fluorescent particles to the antibody or biotin. For example, amidation by reaction of amine and carboxylic acid, sulfidation by reaction of maleimide and thiol, imination by reaction of aldehyde and amine, amination by reaction of epoxy and amine can be used.

(Ii) Labeling agent In immunostaining, first, fluorescent particles are used as a labeling agent.
The “fluorescent particles” according to the present embodiment are fluorescent dye-containing resin particles in which a fluorescent dye is included in a resin.

(Ii-A) Constituent material Examples of fluorescent dyes include rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, Texas Red dye molecules, squarylium dye molecules. And cyanine dye molecules, rhodamine dye molecules, oxazine dye molecules, aromatic ring dye molecules, carbopyronine dye molecules, and the like.
Specifically, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alex 3 Fluor 750, BODIPY FL, BOD 50Y / 568, BODIPY 564/570, BODIPY 576/589 BODIPY 581/591, BODIPY 630/650, BODIPY 650/665 (manufactured by Invitrogen), Cy5, Cy5.5, 1,3-Bis [4- (dimethylamino) -2-hydroxyphenyldiethylenediphenyldienedihydidiethylene , Bis, 1,3-Bis [4- (dimethylamino) phenyl] -2,4-dihydroxycyclobutenedidium dihydride, bis, 2- (4- (Diethylamino) -2-hydroxyphenyl) -4- (4- (diethyl) -2) -Hydroxycyclohexa-2,5-diylenedene)- -Oxocyclobut-1-enolate, 2- (4- (Dibutylamino) -2-hydroxyphenyl) -4- (4- (dibutyliminio) -2-hydroxycyclohexa-2,5-diylenedene) -3-oxocyclobut-1-enolate -(8-Hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido [3,2,1-ij] quinolin-9-yl) -4- (8- hydroxy-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyrido [3,2,1-ij] quinolinium-9 (5H) -ylidene) -3-oxocyc obut-1-enolate, 1-Butyl-2- [5- (1-butyl-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene) -penta-1,3-dienyl]- 3,3-dimethyl-3 eiti-indolium hexafluorophosphate, 1-Butyl-2- [5- (1-butyyl-3,3-dimethyl-1,3-dihydro-indol-2-ylidene) -3-chloro-penta- 1,3-dienyl] -3,3-dimethyl-3H-indolelium hexafluorophosphate, 3-Ethyl-2- [5- (3-ethyl-3H-benzothiazol-2-ylidene) -p nta-1,3-dienyl] -benzothiazol-3-ium iodide, N, N-Di- (2,6-diisopropylphenyl) -1, 6, 7, 12- (4-tert.butyl-phenoxy) -perylen- 3, 4, 9, 10-tetracarbonacid diimide, N, N-Bis (2,6-diisopropylphenyl) -1,6,7,12-tetraphenoxyperylene-3,4: 9,10-tetracarboxdiimide, N, N'-Bis (2,6-diisopropylphenyl) perylene-3,4: 9,10-bis (dicarbimide), Benzenesulfonic acid, 4,4 ', 4'',4'''-[[2,9-bis [2,6 -bis (1-methylethyl) phenyl] -1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra [2,1,9-def: 6,5,10-d 'e'f'] diisoquinoline-5,6,12,13-tetrayl] tetrakis (oxy)] tetrakis-, Benzeneethanaminium, 4,4', 4 '', 4 '''-[[2,9-bis [2 , 6-bis (1-
methylethyl) phenyl] -1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra [2,1,9-def: 6,5,10-d'e'f '] diisoquinoline-5,6,12,13-tetrayl] tetrakis (oxy)] tetrakis [N, N, N-trimethyl-, ROX (X-Rhodamine, Rhodamine Red X), DY-590, 5-ROX, Spectrum Red, PYRROMETHENE650, Texas Red, BODIPY TR, DyLight 594, AlexaFluor 594, HiLyte594, HiLyteFluor TR, Cresyl violet, ATTO590, MFP590, DY-610, ATTO610, DY-615, Oxazine170, ATTO620, C-Phycocyanin, AlexaFluor 633, P633 , DY-630, DY-632, DY-633, MFP631, DyLight633, NorthernLights637, DY-631, DY-634, Nile Blue, APC (Allophycocyanin), APC-XL, EVOblue30, SRfluor 680-CarboxylateLD700 PERCHLORATE, ATTO 655, etc. Can be mentioned.
These fluorescent dyes may be used alone or as a mixture of plural kinds.

Examples of encapsulating resins include polystyrene, polyamide, polylactic acid, polyacrylonitrile, polyglycidyl methacrylate, polymelamine, polyurea, polybenzoguanamine, polyfuran, polyxylene, and phenol resin, which can stably encapsulate fluorescent dyes. Is used.
For the encapsulation of fluorescent dyes, any method of introducing fluorescent dyes into a resin, such as a method of synthesizing particles by binding fluorescent dye molecules to monomers as raw materials, or a method of adsorbing and introducing fluorescent dyes into resin May be used.

(Ii-B) Characteristics such as average particle diameter Next, in immunostaining, two or more kinds of fluorescent particles having different average particle diameters are used. “Average particle size” means that a scanning electron microscope (SEM) is used to take an electron micrograph to measure the cross-sectional area of a sufficient number of particles (1000 particles in this embodiment), and each measured value is a circle. Arithmetic average of the diameter of the circle when the area is taken.
Among these fluorescent particles, fluorescent dye-encapsulating resin particles are used, and the fluorescent dyes may be different or the same, and the encapsulating resins may be different or the same.

As fluorescent particles having a large average particle size (large fluorescent particles), PI dye-containing polymelamine particles containing perylene diimide (PI) in polymelamine, or PI dye-containing polystyrene particles containing PI in polystyrene More preferably, PI dye-containing polymelamine particles are used. From the viewpoint of luminance necessary for HE staining, the average particle diameter of the large fluorescent particles is preferably 120-180 nm (120 nm or more and 180 nm or less).
As the fluorescent particles having a small average particle diameter (small fluorescent particles), Cy5 dye-encapsulated polymelamine particles in which Cy5 is encapsulated in polymelamine, and Cy5 dye-encapsulated polystyrene particles in which Cy5 is encapsulated in polystyrene are used. Preferably, Cy5 dye-encapsulated polymelamine particles are used. From the relationship with the average particle diameter of the large fluorescent particles, the average particle diameter (upper limit) of the small fluorescent particles is preferably 80 nm or less. In the general-purpose microscope, the average particle diameter (lower limit value) of the small-diameter fluorescent particles is preferably 20 nm or more from the viewpoint that it is necessary to be counted as a bright spot at the time of HE staining.

  When producing fluorescent particles with different average particle sizes (particle size control), the amount of encapsulated resin and the amount of fluorescent dye at the time of synthesis are fixed, and the amount of resin is increased or decreased to obtain fluorescent particles with different average particle sizes. It can be synthesized. For example, FIG. 1 shows an example of the distribution of the average particle diameter of fluorescent particles when the ratio of the amount of resin for encapsulation and the amount of fluorescent dye is 0.04.

(Ii-C) Excitation / emission wavelength
Eosin used for HE staining emits fluorescence depending on the microscopic observation conditions. Since the eosin absorption wavelength overlaps with the excitation wavelength of many fluorescent labels, there is a problem that the emission of eosin used for HE staining can interfere with the observation of the fluorescent label.
The fluorescence spectrum (excitation wavelength 520 nm) and excitation spectrum (fluorescence wavelength 540 nm) of eosin are shown in FIG. From the excitation spectrum, it can be seen that eosin is efficiently excited in the wavelength region below 350 nm and in the wavelength region above 450 nm and below 550 nm.
Therefore, the fluorescent particles according to the present embodiment need to be excited by light having a wavelength range of 350 to 450 nm avoiding this wavelength range or a long wavelength range of 550 nm or more.
Preferably, a fluorescent dye that is excited by light having a wavelength of about 580 nm is used for the fluorescent dye of the large diameter fluorescent particle, and a fluorescent dye that is excited by light having a wavelength of 600 nm or more is used for the fluorescent dye of the small diameter fluorescent particle.

[Morphological observation staining]
Among morphological observation stains, especially for tissue specimens, HE staining using two dyes, hematoxylin and eosin, is standardly used as a morphological observation stain for observing the morphology of the specimen, but it is limited to this. It is not something. Examples of other morphological observation staining include Papanicolaou staining (Pap staining) used for cytology.
In HE staining, the nucleus, lime, cartilage tissue, bacteria, and mucus are stained blue-blue to light blue by H staining, and cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes are red by E staining. Although it is dyed dark red, it is not limited to this. Cell nucleus, lime, cartilage tissue, bacteria, and mucus are stained blue-blue to light blue with a dye having the same absorption wavelength as that of hematoxylin analog or hematoxylin, and eosin analog or a dye having an absorption wavelength similar to that of eosin. Cytoplasm, stroma, various fibers, erythrocytes, and keratinocytes may be stained red to deep red.

According to the present embodiment described above, after immunostaining using large-diameter fluorescent particles, immunostaining is performed using small-diameter fluorescent particles, and therefore, staining of a portion that cannot be stained due to steric hindrance of the large-diameter fluorescent particles is performed. (See FIG. 3). In such a case, since the excitation wavelength and emission wavelength of the small-diameter fluorescent particles are 600 nm or more, even if the brightness of the bright spot is low, the bright spot (antigen) can be recognized without being affected by the fluorescence of eosin.
Therefore, it is possible to recognize more antigens even in pathological sections where the number of antigens is large and the steric hindrance of fluorescent particles becomes a problem while maintaining the visibility in HE staining. Even when observation staining is performed together, it is possible to improve the antigen quantification by fluorescent immunostaining.

(1) Preparation of Fluorescent Particle / Labeled Body Sample PI dye-encapsulated polymelamine particles as fluorescent particles and PI dye-encapsulated polymelamine particles bound with streptavidin as labeled bodies were prepared as follows.
N, N′-Bis (2,6-diisopropylphenyl) -1,6,7,12-tetraphenylperylene-3,4: 9,10-tetracarboxdiimide was treated with concentrated sulfuric acid to produce a perylene diimide sulfonic acid derivative. This was converted to an acid chloride to obtain a perylene diimide sulfonic acid chloride derivative.
14.4 mg of perylene diimide sulfonic acid chloride derivative was added to 22.5 mL of water, and then heated on a hot stirrer at 70 ° C. for 20 minutes to give 0.65 g of melamine resin Nicalak MX-035 (Nippon Carbide Industries, Ltd.) 150 nm), and the mixture was further heated and stirred for 5 minutes. After adding 100 μL of formic acid and heating and stirring at 60 ° C. for 20 minutes, the mixture was allowed to cool to room temperature. After cooling, the reaction mixture was placed in a centrifuge tube and placed in a centrifuge for 20 minutes at 12,000 rpm, and the supernatant was removed. This washing was performed with ethanol and water.
0.1 mg of the obtained particles are dispersed in 1.5 mL of EtOH (ethanol), and 2 μL of aminepropyltrimethoxysilane LS-3150 (manufactured by Shin-Etsu Chemical Co., Ltd.) is added and reacted for 8 hours for surface amination treatment. It was.
The obtained dye-encapsulated nanoparticles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. Washing by the same procedure was performed three times to obtain fluorescent dye-containing resin particles (fluorescent particles) having a maleimide group at the end.
On the other hand, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) can be bound to fluorescent dye-encapsulated resin particles after performing thiol group addition treatment using N-succinimidyl S-acetylthioacetate (SATA), followed by filtration through a gel filtration column. A streptavidin solution was obtained.
The fluorescent dye-containing resin particles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. After the obtained solution was concentrated with a centrifugal filter, unreacted streptavidin and the like were removed using a gel filtration column for purification to obtain streptavidin-binding PI dye-encapsulated polymelamine particles A (labeled body).
In such a production method, the amount of polymelamine was increased or decreased with the amount of PI and the amount of polymelamine kept constant, and samples B to H of streptavidin-binding PI dye-encapsulated polymelamine particles were prepared (see Table 1).

(2) Staining method Cultured cells whose total protein amount was previously measured by ELISA were used as pathological sections. HER2 was used as the antigen.
Thereafter, the pathological section was immunostained using Samples A to H, and then morphological observation staining (H staining and HE staining) was performed. Immunostaining and morphological observation staining were performed as follows.
The cultured cells were deparaffinized and then washed with water. The washed cultured cell slide was autoclaved at 121 ° C. for 5 minutes in 10 mM citrate buffer (pH 6.0) to inactivate the antigen.
The cultured cell slide after the inactivation treatment was washed with PBS buffer, and then subjected to blocking treatment with 1% BSA-containing PBS buffer for 1 hour in a wet box. After blocking treatment, anti-HER2 rabbit monoclonal antibody (4B5) (manufactured by Ventana) diluted to 0.05 nM with PBS buffer containing 1% BSA was reacted with the tissue section for 2 hours. This was washed with PBS buffer, and reacted with a biotin-labeled anti-rabbit monoclonal antibody that binds to 4B5 diluted to 2 μg / mL with PBS buffer containing 1% BSA for 30 minutes. After this reaction, the above-mentioned fluorescent dye-containing resin particles diluted to 0.2 nM with PBS buffer containing 1% BSA were subjected to a tissue section, a neutral pH environment (pH 6.9 to 7.4), and room temperature conditions. The reaction was performed for 3 hours. When reacting small-diameter particles, after the above reaction, the cultured cell slide was washed with PBS buffer, and then diluted in 0.2 nM with 1% BSA-containing PBS buffer in a wet box. Reacted. The cultured cell slide was then washed with PBS buffer.
After immunostaining, morphological observation staining (H staining and HE staining) was performed. The immunostained cultured cell slide was stained with Mayer's hematoxylin solution for 5 minutes to perform hematoxylin staining (H staining). The sections were then washed with running water at 45 ° C. for 3 minutes. Next, eosin staining was performed by staining with 1% eosin solution for 5 minutes (HE staining).
After H staining or HE staining, an operation of immersing in pure ethanol for 5 minutes was performed 4 times to perform washing and dehydration. Subsequently, the operation of immersing in xylene for 5 minutes was carried out 4 times to perform clearing. Finally, the tissue section was encapsulated using an encapsulant (“Enterlan New”, manufactured by Merck) to prepare a sample slide for observation.

(3) Counting the number of bright spots The number of bright spots after H staining and the number of bright spots after HE staining were counted as follows.
The tissue sections subjected to immunostaining and morphological observation staining were irradiated with predetermined excitation light to emit fluorescence. The tissue section in that state was observed and imaged with a fluorescence microscope (BX-53, Olympus). The bright spot was measured by the ImageJ FindMaxima method (exposure time was 400 ms and NoiseTolerance was 60). The said excitation light was set to 575-600 nm with respect to PI pigment | dye inclusion polymelamine particle | grains by letting it pass through an optical filter. Moreover, the range of the wavelength (nm) of the fluorescence to be observed was set to 612 to 682 nm by passing through an optical filter. The excitation wavelength condition for microscopic observation and image acquisition was such that the irradiation energy near the center of the visual field was 900 W / cm ² . The exposure time at the time of image acquisition was arbitrarily set (for example, set to 400 milliseconds) so as not to saturate the brightness of the image.
Table 1 shows the results of counting the number of bright spots after H staining and the number of bright spots after HE staining.

(4) Summary As shown in Table 1, paying attention to the number of bright spots at the time of H staining and confirming sequentially from sample A to sample H, the number of bright spots is significantly reduced from sample F to sample G. From this result, it is determined that the average particle diameter (lower limit value) of the fluorescent particles is preferably 20 nm or more in quantifying the antigen using the small-diameter fluorescent particles.
On the other hand, paying attention to the number of bright spots at the time of H staining and the number of bright spots at the time of HE staining, when the difference in the number of bright spots from sample A to sample H is confirmed for each sample, the difference in the number of bright spots in sample D becomes remarkably large. ing. From this result, it is determined that the average particle diameter (upper limit) of the fluorescent particles is preferably 80 nm or less in quantifying the antigen using the small fluorescent particles.

(1) Preparation of fluorescent particle / labeled body sample As shown in Tables 2 to 5, samples of streptavidin-binding fluorescent dye-containing resin particles containing large fluorescent particles or small fluorescent particles were prepared.
In samples 1-1, 2-1, 3-1, and 4-1, commercially available (manufactured by Invitrogen) streptavidin-conjugated Qdot 655 was used.
In Samples 1-2 to 1-11, 2-2 to 2-11, 3-2 to 3-11, and 4-2 to 4-11, labeled bodies were produced in the same manner as in [Example 1]. As in [Example 1], a sample was prepared using PI as the first label used in immunostaining, and the sample was prepared by simply changing PI to Cy5 as the second label used (average particle size). The diameter was controlled by increasing / decreasing the amount of polymelamine while keeping the amount of Cy5 and the amount of polymelamine constant.

In Samples 1-12 to 1-14, 2-12 to 2-14, 3-12 to 3-14, and 4-12 to 4-14, as described below, PI dye-containing polystyrene particles were labeled as fluorescent particles. As above, PI dye-encapsulated polystyrene particles bound with streptavidin were prepared.
PI dye-containing polystyrene particles were prepared by a soap-free emulsion polymerization method. The PI fluorescent dye of [Example 1] was mixed with 4-aminostyrene (manufactured by Tokyo Kasei Kogyo Co., Ltd.) for 1 hour at room temperature to prepare dye-bound styrene. 0.18 g of glycidyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.05 g of styrene (manufactured by Wako Pure Chemical Industries, Ltd.), 0.05 g of divinylbenzene, and 0.005 g of the above dye-bound styrene were added to 5 mL of pure water subjected to argon bubbling. While stirring, the temperature was raised to 70 ° C., 0.012 g of water-soluble azo polymerization initiator V-50 (Wako Pure Chemical Industries, Ltd.) was added and reacted for 12 hours. The reaction solution was centrifuged at 10,000 G for 20 minutes to collect particles. Purification was performed by dispersing the collected particles in pure water and collecting them again by centrifugation. The obtained particles were added to excess ammonia water to convert the epoxy group at the end of the particle into an amino group, thereby obtaining dye-encapsulated polystyrene nanoparticles having an amino group at the end.
The obtained dye-encapsulated nanoparticles were adjusted to 3 nM using PBS (phosphate buffered saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid), and SM (PEG) was added to this solution to a final concentration of 10 mM. ) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleidopropionamid) -dodecaethyleneglycol] ester) was mixed and allowed to react for 1 hour. The mixture was centrifuged at 10,000 G for 20 minutes, the supernatant was removed, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. Washing by the same procedure was performed three times to obtain fluorescent dye-containing resin particles (fluorescent particles) having a maleimide group at the end.
On the other hand, streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) can be bound to fluorescent dye-encapsulated resin particles after performing thiol group addition treatment using N-succinimidyl S-acetylthioacetate (SATA), followed by filtration through a gel filtration column. A streptavidin solution was obtained.
The fluorescent dye-containing resin particles and streptavidin were mixed in PBS containing 2 mM EDTA and allowed to react for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. After 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-binding PI dye-encapsulated polystyrene particles (labeled body).
In Samples 1-12 to 1-14, 2-12 to 2-14, 3-12 to 3-14, and 4-12 to 4-14, PI is simply used as a label used second in immunostaining. Samples were prepared by changing to Cy5 (average particle size was controlled by increasing / decreasing the amount of polystyrene while keeping the amount of Cy5 and polystyrene constant).

(2) Staining method and counting of the number of bright spots In the same manner as in [Example 1], immunostaining and morphological observation staining were performed, and the number of bright spots after H staining and the number of bright spots after HE staining were counted.
In immunostaining, large-diameter fluorescent particles (PI dye-containing resin particles) were reacted, then washed three times with PBS, and small-diameter fluorescent particles (Cy5 dye-containing resin particles) were reacted. In counting the number of bright spots, the excitation light is passed through an optical filter to set 575 to 600 nm and 635 to 650 nm for the PI and Cy5 dye-containing resin particles, respectively, and the range of the wavelength (nm) of the fluorescence to be observed Also, the film was passed through an optical filter and set to 612 to 682 nm and 650 to 700 nm, respectively.
The results of counting the number of bright spots after H staining and the number of bright spots after HE staining are shown in Tables 2 to 5.

  Table 2 shows the total HER2 protein amount by ELISA measurement of 36.1 μg / ml, Table 3 shows the total HER2 protein amount by ELISA measurement of 78.0 μg / ml, and Table 4 shows the total HER2 protein amount by ELISA measurement of 150.8 Table 5 shows the results of μg / ml, and Table 5 shows the results of total HER2 protein amount of 701.8 μg / ml as measured by ELISA.

As shown in Tables 2 to 5 and FIG. 4, Samples 1-1, 2-1, 3-1, and 4-1 were immunostained only with a small-diameter labeling agent (Qdot655), and the brightness at H staining was high. The score is considered to be a more accurate value for quantification of the antigen, but since the brightness of the bright spot itself is low, the bright spot number is drastically reduced due to the influence of eosin fluorescence during HE staining.
In Tables 2 to 5, the number of bright spots at the time of H staining in Samples 1-1, 2-1, 3-1, 4-1 is a more accurate value and ideal value for quantification of the antigen. It can be determined that the closer the total number of bright spots is to this value, the better the antigen quantification.
In Samples 1-2, 2-2, 3-2, and 4-2, immunostaining was first performed with small-diameter fluorescent particles, followed by immunostaining with large-diameter fluorescent particles. Therefore, it cannot be said that the quantification of the antigen is improved.

On the other hand, in samples 1-3 to 1-11, 2-3 to 2-11, 3-3 to 3-11, 4-3 to 4-11, immunostaining was first performed with large fluorescent particles, and then Immunostaining is performed with small-diameter fluorescent particles, and the number of bright spots due to the small-diameter fluorescent particles is large at the time of HE staining, and it can be said that quantification of the antigen is improved.
From these results, when both fluorescent staining and morphological observation staining are performed, in order to improve antigen quantification by fluorescent staining, immunostaining is first performed with large fluorescent particles, and then immunostained with small fluorescent particles. It turns out that is useful.
Furthermore, Samples 1-6 to 1-8, 2-6 to 2-8, 3-6 to 3-8, 4-6 to 4-8, Samples 1-12 to 1-14, and 2-12 to 2-2 -14, 3-12 to 3-14, 4-12 to 4-14, the former sample group has more bright spots due to small-diameter fluorescent particles at the time of HE staining, as a resin for encapsulating fluorescent particles It can be seen that polymelamine is more preferable than polystyrene.

10 Label 12 Fluorescent Particle 14 Biotin 20 Antigen 30 Primary Antibody 40 Secondary Antibody 42 Avidin

Claims (6)

In a staining method of a pathological section using two or more kinds of fluorescent particles having different average particle diameters, which are fluorescent particles encapsulating a fluorescent dye in a resin,
Immunostaining a specific antigen with the first fluorescent particles having a first average particle diameter and emitting a first fluorescent color with respect to the pathological section;
A second fluorescent color having an average particle size smaller than the first average particle size of the first fluorescent particles and different from the first fluorescent color is applied to the pathological section . Immunostaining the specific antigen using second fluorescent particles that emit ;
A staining method characterized by comprising: The staining method according to claim 1, wherein
The staining method, wherein a difference in average particle diameter between the first fluorescent particles and the second fluorescent particles is 40 nm or more. The staining method according to claim 1 or 2,
An average particle diameter of the first fluorescent particles is 120 to 200 nm. In the dyeing | staining method as described in any one of Claims 1-3,
The average particle diameter of the first fluorescent particles is 120 to 200 nm,
The staining method, wherein the average particle diameter of the second fluorescent particles is 20 to 80 nm. In the dyeing | staining method as described in any one of Claims 1-4,
Further comprising the step of staining the pathological section with hematoxylin and eosin,
A staining method characterized in that fluorescent particles excited by light having a wavelength of about 580 nm are used as the first fluorescent particles, and fluorescent particles excited by light having a wavelength of 600 nm or more are used as the second fluorescent particles. . In the dyeing | staining method as described in any one of Claims 1-5,
A staining method comprising performing a step of immunostaining the specific antigen using the second fluorescent particles after the step of immunostaining the specific antigen using the first fluorescent particles .

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