JP3127244B2 - A dual label detection method combining chemiluminescence in situ hybridization and immunohistochemical staining - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a chemiluminescence in s
Itu hybridization (chemiluminescent in
The present invention relates to a double-label detection method that combines in situ hybridization and immunohistochemistry by coloring.

[0002]

2. Description of the Related Art Detection of substances expressed in cells,
Identification of cell phenotype is also an important tool for elucidating the pathological mechanisms of various diseases.

[0003] As a means for detecting substances expressed in cells such as nucleic acids and proteins, mRNA is in situ.
Tu detection methods are widely used. mRNA
Is commonly detected by in situ hybridization with a labeled probe. In recent years, attempts have been made to use such chemiluminescence as an indicator to detect such mRNA, but the in situ hybridization method using a chemiluminescent substance as a label has limited measurement sensitivity.

On the other hand, advances in science have made it necessary to detect minute amounts of disease-related factors and a small number of cells that express them. For example, human T-lymphotropic virus type I-associated myelopathy
For the onset of yelopathy: HAM, HTLV-I (human Tl
ymphotropic virus infection (Mitsuhiro Nori, HTLV-1-associated Myelopathy (HAM), medicina, 1
988 25 (10): 2216-2217). However, very few cells express the HTLV-Itax gene (part of the HTLV-I-specific mRNA sequence) in the peripheral blood of patients who develop HAM, and the expression level of HTLV-I itself is very low. Due to the small number, studies using conventional methods have been very difficult. The first reason is that it is difficult to find a target infected cell in a specimen obtained from a living body. Second, determine the cellular phenotype of the infected cells, even if they could be found, unless a second, different detection method, such as immunohistochemical staining, can be applied to the identified cells themselves. Even that is difficult. Furthermore, since the amount of expressed mRNA is small, measurement can be performed with high sensitivity.
It is limited to the u-hybridization method. Therefore, there is a demand for the development of a detection means that can perform the same cell in combination with a detection method such as a chemiluminescence in situ hybridization method and an immunostaining method, and that has extremely excellent detection sensitivity. It had been.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for performing double label detection in the same specimen by chemiluminescence in situ hybridization and immunohistochemical staining, and to provide a method for detecting these two labels. An object of the present invention is to provide a detection device for obtaining both with high sensitivity and high resolution.

[0006]

Means for Solving the Problems As a result of earnest research, the present inventors have found that the following objects can be solved by the following means and the object can be achieved. That is, chemiluminescence in situ
A double-label detection method for detecting a tissue or a cell having a specific nucleic acid obtained by combining u-hybridization and immunohistochemical staining and having a second cell-related substance other than the nucleic acid, , On tissue sections or cells in the specimen,
Performing chemiluminescence in situ hybridization using a probe for a target intracellular nucleic acid, obtaining a luminescence image after obtaining a transmitted light image of the sample, and storing a position where luminescence occurs in the sample. And then performing immunohistochemical staining using an antibody against the second cell-related substance, and transmitting the transmitted light image of the specimen subjected to the immunohistochemical staining at the memorized position. And a step of obtaining.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for in situ chemiluminescence.
u hybridization (chemiluminescent in situ
hybridization and immunohistochemical staining by color development (im
The present invention discloses a detection method capable of performing the double-label detection method consisting of munohistochemistry) on the same sample and a slide glass suitable for the detection method. Further, the present invention discloses a detection device for obtaining chemiluminescence and color detection with high sensitivity and high resolution. More specifically, by performing in situ hybridization and immunohistochemical staining stepwise on the same cell in the same specimen, two or more pieces of information on the target cell, for example, mRNA
It is possible to obtain information on the expression of, and information on the cell phenotype.

What can be detected in the present invention is in situ
DNA and RNAs in u-hybridization, cell-related substances such as peptides, proteins, hormones, sugar chains, and signal transduction substances in immunohistochemical staining. These can be detected in combination according to the purpose. It is. By detecting these, information on DNA profile, mRNA or protein expression, virus infection, cell phenotype, etc. can be obtained, and biological analysis, physiological analysis, medical analysis, pathological analysis, etc. It can be used in a wide range of research. In particular, mR
The combination of chemiluminescence in situ hybridization for NA and immunohistochemical staining for a cell surface antigen is useful for diagnosis of HAM and the like.

The detection apparatus, detection method, and sample preparation method of the present invention will be described in detail below.

[Detection Apparatus] The apparatus of the present invention uses a chemiluminescence i
By performing two detection methods, one for the n situ hybridization method and the other for the immunohistological detection method using color development, on one sample in a stepwise manner, mRNA expression and cell expression can be performed on one sample, especially on the same cell. This is a device that can analyze the mold.

FIG. 1 is a block diagram showing the structure of the present apparatus. The detection apparatus of the present invention can obtain images at all positions on the target sample, and can store the detected positions and reproduce the stored detected positions.
An epi-fluorescence microscope equipped with a Y-stage, and a color CCD connected to the epi-fluorescence microscope via a double port lens barrel
A device comprising a camera and a monochrome CCD camera capable of detecting chemiluminescence, an image processor connected to the two CCD cameras, a data analyzer connected to the cameras, and a monitor connected to the data analyzer. .

An XY stage that can be used in the apparatus of the present invention can obtain images at all positions on a target sample, and can store a detected position and reproduce the stored detected position. Should be fine. A color CCD camera can be any commonly used color CC
D camera may be used, but color Peltier cooling system 3CCD
A camera is preferred but not limited to this. A monochrome CCD camera is any commonly used color CCD as long as it can detect chemiluminescence.
It may be a camera. In particular, a Peltier-cooled CCD camera or measurement is preferred, but not limited thereto. It is preferable to use in combination such that chemiluminescence is detected by a Peltier-cooled CCD camera and an image by transmitted light is captured by a color-Peltier-cooled 3CCD camera.

[Double-Label Detection Method] The double-label detection method of the present invention is a two-stage detection method for chemiluminescence in situ hybridization and an immunohistological detection method based on color development. In this method, mRNA expression and cell phenotype can be analyzed for one specimen, particularly the same cell.

The outline of the method of the present invention is as shown in the scheme of FIG. First, tissues or cells are collected from humans or experimental animals. The obtained target tissue or cell is fixed on a slide glass, and is pre-treated so that staining is possible. The method of pretreatment can be performed by a method known per se. As a slide glass used for fixing a tissue or a cell, a slide glass with a shallow chamber described later is preferable.

The obtained sample is subjected to in situ hybridization using a labeled oligonucleotide probe (hereinafter, also simply referred to as a labeled probe). Hybridization can be performed by any method known per se. Further, the labeling substance of the present invention includes alkaline phosphatase,
Although it is possible to use enzymes such as peroxidase, alkaline phosphatase is preferred. After completion of the hybridization, the sample is washed and a chemiluminescence reaction is performed.

[0016] Chemiluminescence is performed by adding a chemiluminescent substrate for alkaline phosphatase. The generated chemiluminescence image is captured by a CCD camera, and the position of the cell is stored. As the substrate, any substrate can be used as long as it can obtain chemiluminescence,
It is preferable to use CDP-star (TROPIX), since a light emission amount with a strong immediate effect and long-lasting light emission can be obtained. In the method of the present invention, it is also preferable to use various sensitizers for suppressing quenching of luminescence by water, and it is preferable to use emerald-II (TROPIX).
In addition, the use of CDP-star and Emerald-II together with the presence of polyvinyl alcohol described below makes it possible to stably detect chemiluminescence with high sensitivity and improve the resolution.

Subsequently, the specimen is subjected to immunohistochemical staining, and the color development at the position stored in the previous step is observed. In the present invention, a general immunohistochemical method can be used. In addition, any antibody capable of detecting the antigen of interest and any dye for staining can be used. The labeling substance used for staining may be a coloring dye or a fluorescent substance. Emerald-II
Contains a fluorescent substance. Therefore, when a fluorescent substance is used as a labeling substance, the use of Emerald-II is not preferable.

Further, according to the method of the present invention, immunohistochemical staining is performed by chemiluminescence in s depending on the measurement object and the measurement item.
It can also be performed before itu hybridization.

[Preparation of Specimen] Tissue or cells obtained from test subjects such as patients and experimental animals
After fixing to the slide glass with a shallow chamber shown in FIGS. 2 and 3, observation and detection are performed.

As shown in FIGS. 2 and 3, the slide glass 1 with a shallow chamber includes a slide glass 2, a cover glass 3, a wall 4 located between the slide glass 2 and the cover glass 3, and these elements. It has a chamber 5 formed by being surrounded.

The chamber 5 formed by the slide glass 2, the cover glass 3, and the wall 4 can hold a liquid inside. Therefore, it is desirable that the wall part 4 is fixed to the slide glass 2 and the cover glass 3 in close contact at least at the time of measurement.

The slide glass 2 and the cover glass 3
Commercial products can be used. The size of the cover glass 3 can be selected according to the purpose. The wall 4 is made of glass, resin,
It can be formed of metal or the like. The area surrounded by the wall portion 4 can be selected according to the purpose, and the shape of the surrounded portion may be rectangular or circular.
The chamber 5 has a depth of 0.1 mm to 0.3 mm, preferably 0.2 mm. If the depth is larger than that, it becomes very difficult to focus the microscope, which is not desirable.

After the target specimen is fixed on the slide glass 2, the wall 4 and the cover glass 3 can be installed, or the specimen can be fixed on the slide glass 1 with a shallow chamber formed in advance. It is.

The specimen fixed in the chamber 5 is observed or detected by adding a detection reagent or a staining solution. Since the chamber 5 has a certain volume,
It is possible to hold the liquid. Therefore, observation or measurement can be performed without removing or washing the liquid after treating the staining liquid or the like. Furthermore, even for chemiluminescence, which has conventionally been extremely difficult to obtain high detection sensitivity, by holding the liquid in the chamber 5, the detection sensitivity is improved, and at the same time, high resolution can be obtained. The reason why such an effect can be obtained is that the brightness of the image is increased by holding the liquid, and that the outline of the measurement object can be clearly displayed.

Further, in the conventional technique used for detecting one sample by a plurality of stains, cells or tissues are damaged by repeated staining and washing.
However, by using the slide glass with a shallow chamber of the present invention, the cover glass can be prevented from directly contacting the sample (tissue or cell), so that damage to the cell or tissue due to repetition of the operation can be prevented. It is possible. Further, since the shallow chamber has high hermeticity, it has excellent ability to hold the liquid in the chamber. Therefore, it is possible to prevent the sample from drying for a long time.

2. Polyvinyl alcohol If the above-described slide glass 1 with a thin chamber is used and polyvinyl alcohol is used as described later, it is possible to obtain highly sensitive detection and high resolution.
At the same time, since polyvinyl alcohol is an inexpensive material, the running cost can be reduced by mixing it with various sensitizers conventionally used.

The use of polyvinyl alcohol is suitable for commonly used detection methods such as a chemiluminescence method, a fluorescence method, and a coloring method. In particular, the sensitivity and resolution can be effectively improved by using the composition for the chemiluminescence method as compared with the conventional method.

The polyvinyl alcohol of the present invention preferably uses 13 to 23 kd. Further, it is preferable to use such that the final concentration becomes 10% v / v. Polyvinyl alcohol can give viscosity to the applied liquid. Due to this viscosity, movement of the liquid in the chamber can be suppressed, and uneven diffusion of the dye and the like can be prevented.

On the other hand, when chemiluminescence is detected, a commonly used sensitizer such as emerald-II ^™ (TROPIX) can be used together with polyvinyl alcohol. By using polyvinyl alcohol in combination, the use of expensive sensitizers such as Emerald-II ^™ can be reduced.

Example I. Apparatus The apparatus shown in FIG. 5 was used. The details will be described below.

This apparatus is a Peltier-cooled CCD camera (The Night OWL Molecular Light Imager, EG & G, Berth
old, Bad Wildbad, Germany) and a color-Peltier cooled 3CCD camera (M-3204, Olympus, Tokyo) via a double-port lens barrel, and a laser scanning cytometer (LSC101-H3, Olympus) The epi-fluorescence microscope B × 50F (Olympus) connected.

Further, the detection device of the present invention is a laser scanning cytometer (LS) manufactured by Olympus Optical Industries, Ltd.
It is configured based on a microscope equipped with C101). For more information,
A microscope having an XY stage of LSC101, a color chilled camera and a chilled camera connected to the XY stage using a double port lens barrel, transmitted light installed on the microscope, an image processor connected to the two cameras, and data analysis Computer consisting of devices (The Nigh
t OWL Molecular Light Imager, EG & G, Berthold, Bad
Wildbad, Germany) and the computer has:
An RGB color monitor (Compaq) and a printer are connected. The software is software installed on the computer (The Night OWL Molecular Light Imag
er). The measurement was performed by detecting chemiluminescence with a Peltier cooling CCD camera and using a color Peltier cooling system.
An image due to transmitted light was captured by a CCD camera.

The analysis system of the laser scanning site meta-system includes a computer (IBM PC / PT convertible computer), a 17-inch multi-scan monitor, a color printer, and a power controller.

Laser scanning cytometer LSC1
01 itself uses a laser as excitation light, laser-scans an arbitrary designated area on a slide glass, and quantifies the fluorescence emitted from the specimen, thereby quantitatively determining the biochemical components and the like of the fluorescently stained substance. It is a device for measuring at a time. In addition, this device measures the amount of fluorescence while
Since it has the function of storing the XY coordinates of each substance, it is possible to immediately adjust the position in the specimen corresponding to the coordinates to the range of the field of view of the microscope based on the stored coordinates. A possible device.

The inventors changed the structure of the laser scanning cytometer, detected chemiluminescence, stored the XY coordinates of each substance, and corresponded to the coordinates based on the stored coordinates. A device capable of immediately adjusting the position in the specimen to be measured to within the field of view of the microscope was completed.

II. Measurement 1. Performance of Chemiluminescence Detection Device and Effect of Polyvinyl Alcohol In order to clearly show the performance of the chemiluminescence detection device of the present invention and the effect of polyvinyl alcohol, detection of cell phenotype by immunohistological detection method using chemiluminescence went. Immunohistological detection methods using chemiluminescence are still widely used today. The detection accuracy has been established to some extent. Therefore, by using an immunohistological detection method using chemiluminescence, it is possible to prove the performance of the chemiluminescence detection device of the present invention and the effect of polyvinyl alcohol. Specifically, CD45RO ⁺ T cells were detected by chemiluminescence using an OPD4 antibody on a human tonsil specimen.

Human T-cell leukemia virus type I (human T-
Pathological specimens were prepared from tonsillar tissues collected from patients infected with lymphotropic virus type I) using commonly used methods. This specimen was subjected to immunohistological detection as follows.

An OPD4 antibody (mouse, Dako) that specifically binds to CD45RO ⁺ T cells was used as a primary antibody. OPD4 antibody diluted 1:25 was added to the specimens, incubated, and subsequently washed.

Next, a rabbit anti-mouse immunoglobulin (Dako) diluted 1:25 followed by a 1:50 alkaline phosphatase: anti-alkaline phosphatase (APA)
(AP, Dako) complex (APAAP method) was added to the sample and incubated (Cordell JL, Falini B, Erber WN,
Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KA, St
ein H, Mason DY, Immunoenzymatic labeling of monoc
lonal antibodies using immune complexes of alkalin
e phosphatase and monoclonal anti-alkaline phospha
tase (APAAP complexes). J Histochem Cytochem 1984 F
eb; 32 (2): 219-29).

As a chemiluminescent substrate for alkaline phosphatase, CDP-Star was used. C
By using DP-star, alkaline phosphatase can be optically detected. CDP-star is 1:10 in detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5).
Diluted at 0. During this dilution, if necessary, polyvinyl alcohol (13-23 kd) was added at 10% v / v to increase the viscosity. The mixture for detection was added to the chamber, and a cover glass was provided.

The chemiluminescence in the obtained specimen was detected by accumulating the luminescence for 1 minute using the detection apparatus of the present invention.
FIG. 6 shows the result.

FIGS. 6A and 6B are micrographs of the specimen detected in the presence of 10% polyvinyl alcohol, and FIGS. 6C and 6D are micrographs of the specimen without 10% polyvinyl alcohol (magnification). × 100). A and B show detection by chemiluminescence, and C and D show detection by transmitted light.

In the detection of CD45RO ⁺ T cells in the specimen, the specimen using 10% polyvinyl alcohol
Both spatial resolution and luminescence intensity were excellent, and even individual cells could be detected (FIG. 6-A). On the other hand, the detection in a sample not using 10% polyvinyl alcohol did not provide the resolution at the single cell level.

From the above results, the detection apparatus of the present invention has extremely excellent performance, and further higher performance can be obtained by using polyvinyl alcohol.

3. In situ hybridization HTLV-I producing T cell line (MT-2 cell) and virus-negative B cell line (Raji cell) were transformed with 10% fetal bovine serum, penicillin G (50 units / ml) and streptomycin (50 μ / ml). ) Was cultured in RPMI1640 medium. After washing the cells with phosphate buffered saline (PBS) -CMF, the obtained cell suspension was dropped on a silane-coated slide glass (Perkin Elmer) and fixed with 4% paraformaldehyde for 30 minutes.

Subsequently, a shallow chamber was formed in the obtained specimen. The height of the wall is 0.2mm and the area is 250m
As will be m ^2, and to form a shallow chamber by adhering a double-sided adhesive tape on the glass slide.

As a probe in this test, amino-modified deoxythymidine triphosphate (dTTP) introduced solely during DNA synthesis was added to an amine-reactive divalent cross-linking agent, disuccinimidyl suberate.
uberate; DSS) was used to bind the oligonucleotide to alkaline phosphatase. The obtained labeled oligonucleotide was purified by high performance liquid chromatography (HPLC) (Jablonski E, Moomaw EW,
Tullis RH, Ruth JL, Preparation of oligodeoxynucle
otide-alkaline phosphatase conjugates and their us
e as hybridization probes.Nucleic Acids Res 1986
Aug 11; 14 (15): 61115-28, Ruth JL, Morgan C, Pasko A,
Linker arm nucleotide analogs in oligonucleotide
synthesis.DNA 1985; 4:93, Kiyama H, Emson PC, Tohy
ama M, Recent progress in theuse of the technique
of non-radioactive in situ hybridization histochem
istry: new tools for molecular neurobiology. Neuro
sci Res (NY) 1990 Sep; 9 (1): 1-21). In addition, the probe has a sequence of HTLV-Itax region: 5 ′ ACG CCC TACTGG
CCA CCT GTC CAG AGC ATC AGA TCA CCT G 3 '(7425-746
The sense and antisense strands with (4) were used (SeiK
i M, Hattori S, Hirayama Y, Yoshida M, Human adult
T-cell leukemia virus: complete nucleotide sequen
ce of the provirus genome integrated in leukemia c
ell DNA. Proc Natl Acad Sci USA 1983 Jun; 80 (12):
3618-22). The antisense oligonucleotide probe labeled dTTP at position 13 and the sense probe labeled dTTP at position 29. In addition, the gag region of human immunodeficiency virus (HIV) was used as a control.

To detect tax messenger RNA (mRNA) by in situ hybridization, cells were treated with 0.2N hydrochloric acid for 20 minutes and 5 μg
/ Ml proteinase K at 37 ° C. for 15 minutes and then acetylated. Hybridization is
10 fmol / ml of 10% deionized formamide, 10% dextran sulfate, 4 times standard citrate saline (hereinafter referred to as SSC buffer), 400 μg / ml salmon sperm DNA, and 1 time Denhardt's solution (Denhardt's)
μl of probe was annealed in a humid chamber at 37 ° C. overnight. After washing in 55 ° C., 1 × SSC buffer, the bound probe was washed with C in detection buffer containing 10% polyvinyl alcohol (13-23 kd).
Detected by DP-star. Furthermore, a buffer solution in which emerald-II was further added to the substrate buffer at a concentration of 10% and a buffer solution in which emerald-II was not added were prepared, and the effects of emerald-II were compared. For the obtained specimen, images of transmitted light and chemiluminescence were obtained by the method described above. The results are shown in FIG.

FIGS. 7A to 7F are images obtained by the present detection apparatus. More specifically, FIGS. 7A, 7C, and 7E are images detected by chemiluminescence, and FIGS. 7B, 7D, and 7F are images detected by transmitted light (magnification × 200). 7A-D are MT-2 cells producing HTLV-I. As is evident from the figure, the cells produced high intensity chemiluminescence. In contrast, FIGS. 7E and F
Is an image obtained from a specimen using Raji cells that do not produce HTLV-I. Raji cells are HTL
Chemiluminescence did not occur even after hybridization with the V-Itax antisense probe. In addition, chemiluminescence did not occur in MT-2 cells following hybridization to the sense or HIV-specific probes (not shown).

Therefore, by using the detection device of the present invention,
Only the target mRNA can be reliably detected.

FIG. 7A is an image of a specimen to which Emerald-II is added, and FIG. 7C is an image of a specimen to which no emerald-II is added. In FIG. 7-A, the emission signal was enhanced and the background was suitable for measurement, whereas in FIG. 7-C, the background emission was high and the emission signal was also increased. I couldn't. Therefore, when the device of the present invention is used, Emerald-II
Is more preferred.

4. Double label detection of in situ hybridization and immunohistochemical staining To demonstrate the efficacy of the dual label detection method of the present invention, determine the cell phenotype of cells capable of detecting viral mRNA. did.

The cells used in this test were MT-2 cells, which are HTLV-I producing T cell lines, and virus-negative B cell lines (Raji cells). The probe used was HTL.
V-Itax antisense probe. The cells were first incubated with 3% hydrogen peroxide in methanol, followed by pretreatment, hybridization and washing, followed by detection of chemiluminescence as described above. Subsequently, the cells are incubated with an antibody against OPD4 (previously diluted as already described),
Peroxidase-labeled polymer (Dako En
vision System). In addition, horseradish peroxidase (HR)
As a substrate for P), 3-amino-9-ethylcarbazole (AEC) was used. The position of the cell where the chemiluminescence was positive was reproduced by the XY stage, and a color image by transmitted light was obtained by a color Peltier cooling 3CCD camera. FIG. 8 shows the result.

FIG. 8A is a photograph showing the detection of chemiluminescence in which the chemiluminescence is represented by a pseudo blue color, and FIG.
It is the photograph detected by the transmitted light which developed red by dyeing | staining of C. The cells indicated by the arrow in the center are the virus-positive cells of interest. In FIG. 8-A, the target cells exhibited blue, and the other cells did not exhibit blue. In FIG. 8B as well, the target cells exhibited red, and the other cells did not exhibit red.

Therefore, at the position of the chemiluminescence generated by the virus-positive cells, the image of the chemiluminescence represented by the pseudo blue color (FIG. 8-A) and the transmitted light image (FIG. 8-B) are superimposed. In the sample subjected to the double labeling detection method, it was confirmed that the cell phenotype of all cells in which viral mRNA was detected was CD45RO ⁺ T cells.

The use of the dual-label detection method of the present invention has demonstrated that chemiluminescence in situ hybridization and immunohistochemical staining can be performed with high sensitivity and high resolution. The position reproducibility with a laser scanning cytometer was +/− 5 μm.

[0057]

According to the detection device and the detection method of the present invention,
It is possible to perform a double-label detection method using chemiluminescence in situ hybridization and immunohistochemical staining on the same specimen, and the images obtained by the chemiluminescence and transmitted light obtained by the detection apparatus of the present invention are both highly sensitive. And high resolution.

In particular, according to the sample preparation method of the present invention using a slide glass with a shallow chamber and polyvinyl alcohol, an image detected by chemiluminescence and transmitted light can be obtained with higher sensitivity and higher resolution.

It is possible to detect a small amount of mRNA expressed in a small number of infected cells with high sensitivity, such as detection of the HTLV-Itax gene in HAM patients and analysis of the cell phenotype of infected cells obtained from the patients. It is possible, and furthermore, the cell phenotype of the detected infected cells can be easily determined.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a detection device of the present invention.

FIG. 2 is a plan view of a slide glass with a shallow chamber.

FIG. 3 is a cross-sectional view of the slide glass with a shallow chamber of FIG. 1 taken along line AA ′.

FIG. 4 is a scheme diagram showing the double label detection method of the present invention.

FIG. 5 is a schematic diagram showing an embodiment of the detection device of the present invention.

FIG. 6. Anti-OPD4 antibody (CD4
5RO ⁺ T cells) are micrographs showing images of chemiluminescence in immunohistochemistry by chemiluminescence.

FIG. 7: HTLV-I tax mRNA using an alkaline phosphatase-labeled oligonucleotide probe
5 is a micrograph showing an image of chemiluminescence by in situ hybridization of the chemiluminescence of FIG.

FIG. 8 is a micrograph showing an image obtained by double labeling in situ hybridization.

[Explanation of symbols]

 1. Slide glass with shallow chamber 2. Slide glass 3. Cover glass 4. Wall 5. Chamber

Continuation of the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/536 C12N 15/09 C12Q 1/68 G01N 33/566 G02B 21/34

Claims (3)

(57) [Claims] 1. A method for detecting cells expressing the HTLV-Itax gene in a sample obtained from a patient infected with human T-cell leukemia virus type I, and determining the cell phenotype of the cells. (1) performing a chemiluminescence in situ hybridization on a tissue section or a cell in a specimen using a probe for the gene; and (2) obtaining a luminescence image after obtaining a transmitted light image of the specimen. (3) a step of storing a position where light emission occurs in the specimen; (4) a step of performing immunohistochemical staining using an antibody against a cell-related substance; and (5) the storage Obtaining a transmitted light image of the specimen that has been subjected to the immunohistochemical staining at the designated position. 2. The method according to claim 1, wherein the steps (2) to (5) are performed as follows: (1) Images can be obtained for all positions on a target specimen. An epi-fluorescence microscope equipped with an XY stage capable of storing the detected position and reproducing the stored detected position; (2) the two CCD cameras; and (3) data analysis connected to the camera. And (4) a monitor analysis device connected to the data analysis device. 3. The method according to claim 1, wherein the tissue section or the cell is fixed in a chamber provided on a slide glass to be a specimen, and the luminescent image is obtained. The method, wherein the sample in the step of obtaining is provided with a staining solution containing polyvinyl alcohol.