JP2011188835A - Tissue microarray and tissue analysis method - Google Patents

Abstract

Provided is a tissue microarray that easily obtains a comprehensive protein expression profile of an individual patient and enables personalized medicine in a clinical field.
A tissue microarray in which a plurality of small sliced tissue sections 100 of the same tissue of the same case of the same person are arranged on a slide plate 200. On the slide plate 200, a positive control 110, a negative control 120, and a normal tissue 130 can be arranged in addition to a plurality of cut thin sliced tissue sections 100. The cut sliced tissue sections 100 of the tissue microarray 900 are each stained with a plurality of staining markers.
[Selection] Figure 1

Description

  The present invention relates to a tissue microarray (TMA) used in medical research and pathological diagnosis, and a tissue analysis method using the tissue microarray.

  Many techniques for analyzing biological materials have been developed. A microarray technique is often used as a technique for analyzing biological materials. For example, a DNA microarray is obtained by fixing thousands of DNA probes on a substrate such as a slide glass. A sample labeled with a fluorescent molecule or the like is flowed and hybridized with a DNA probe, and the intensity of fluorescence emission is measured to estimate the expression level of a gene contained in the sample.

  Immunohistochemistry is a method of analyzing a specific gene expression protein using an antibody that recognizes the expressed protein. In situ hybridization is a method of analyzing gene expression on a tissue using a primer complementary to the base sequence of the target gene. Both immunohistochemistry and in situ hybridization can be analyzed using paraffin blocks.

  The tissue microarray has a large number of tissues placed on one slide plate. For example, 100 to 1000 small tissue pieces are placed on the slide plate, and these tissues can be stained and analyzed.

  In immunohistochemistry and in situ hybridization, one tissue section is placed on one slide, but by using a tissue microarray, a large number of tissue sections can be placed on one slide, saving labor. Rapid analysis and research will be possible.

  For example, Patent Document 1 discloses that test tissues of different cases are hollowed out from a paraffin block into a cylindrical shape having a diameter of 0.6 to 2 mm, and a large number of hollowed test tissues are collected and embedded in a new paraffin block. Then, a tissue microarray is described, which is formed by slicing and then placing a sheet-like slice section sample on which a tissue section is placed on a slide glass. Then, comprehensive analysis is performed by staining a large number of tissue sections on the slide plate with a single marker.

  Further, for example, in Patent Document 2, a tissue to be examined is hollowed out from an organ-fixed paraffin block of different cases with a cylindrical blade into a cylindrical shape, and a plurality of hollow holes having the same diameter as the cylindrical blade are formed in advance. A tissue microarray is prepared by preparing a tissue block, embedding a hollow test tissue in a hollow hole of the tissue block, pouring a paraffin solution between the embedding dish and the tissue block, and filling and solidifying the tissue block. .

  Further, for example, in Patent Document 3, a paraffin support block having a plurality of holes is prepared, and a test tissue having a shape corresponding to the shape of the hole is collected from a sample tissue block of a different case embedded in paraffin. A tissue microarray is described in which the test tissue is inserted into the hole of the support block and the support block is heated to melt the paraffin so that the support block and the test tissue are integrated together. .

JP 2005-530504 Gazette JP 2004-258017 A JP 2006-162489 A

  However, a conventional tissue microarray has a large number of tissue sections of different cases from different patients placed on a slide plate. Tissue analysis using the tissue microarray can be performed using a single marker for these tissue sections on the slide plate. It is to be dyed. Therefore, a comprehensive protein expression profile cannot be obtained for each patient, and personalized medicine in the clinical field is extremely difficult.

  The present invention has been made in view of such problems, and it is possible to easily obtain a comprehensive protein expression profile of an individual patient and to enable individualized medical treatment in a clinical field, and the tissue microarray. An object of the present invention is to provide a tissue analysis method using the

  The tissue microarray according to the first aspect of the present invention is characterized in that a plurality of small sliced tissue sections of the same tissue of the same case of the same person are arranged on a slide plate.

  In addition to a plurality of small sliced tissue sections, it is preferable to place a separate positive control for each marker on the slide plate.

  In addition to a plurality of small sliced tissue sections, it is preferable to place a separate negative control for each marker on the slide plate.

  Further, in addition to a plurality of small sliced tissue sections, it is preferable to arrange the same case with respect to the target tissue or normal tissues of other cases on the slide plate. For example, in the case of breast cancer tissue, it is normal breast tissue of the same case.

  Moreover, it is preferable that a tissue slice placement site indicating a site where the small sliced tissue slice is placed is described on the slide plate.

  Further, it is preferable that a name of a staining marker for staining the sliced tissue section is described on the slide plate.

  Moreover, it is preferable that the name of the antigen activation solution suitable for the staining marker for staining the small sliced tissue section is described on the slide plate.

  Moreover, it is preferable that the name of the secondary antibody suitable for the primary antibody of the staining marker for staining the small sliced tissue section is described on the slide plate.

  Furthermore, the description of the tissue section placement site, the staining marker name, the antigen activation solution, or the name of the secondary antibody is preferably described by laser marking on the slide plate.

  In the tissue analysis method according to the second aspect of the present invention, a plurality of small sliced tissue sections of the same tissue of the same person of the same person are arranged on a slide plate, and the sliced tissue sections are stained with a plurality of staining markers. It is characterized by dyeing each.

  Further, on the slide plate, a tissue staining sheet having a plurality of through holes provided in a base material is placed, and an antigen activation solution suitable for each staining marker is placed in the plurality of through holes of the tissue staining sheet, a plurality of It is preferable to inject and stain each of the staining markers and secondary antibodies suitable for the primary antibody of each staining marker.

  In the tissue microarray of the present invention, a plurality of small sliced tissue sections of the same tissue of the same person in the same case are arranged on the slide plate, and each of the sliced tissue sections is stained simultaneously with a plurality of staining markers, Comprehensive protein expression profiles of individual patients can be easily obtained, enabling personalized medicine in clinical settings. Therefore, the patient's QOL (Quality of Life) is achieved by maximizing the main effects and minimizing side effects based on the patient's individual molecular / genetic information and information on the molecular / genetic abnormalities that form the cause and pathology of the disease. It is possible to formulate and execute a treatment plan that optimizes. Furthermore, by simultaneously staining the tissue microarray of the present invention using a number of staining markers, a series of information transmission until the cell receives a stimulus from the outside via a receptor on the cell surface and the function of the cell changes. Clinically effective knowledge about the process (signal transduction) can also be obtained. Furthermore, the amount of antibody during the incubation can be small, and the number of slide plates can be reduced.

1 is a perspective external view of a tissue microarray according to the present embodiment. It is a perspective appearance figure of a slide plate in which a tissue section arrangement site and a staining marker name are described. It is a perspective external view of the slide plate in which the name of the staining marker is described in the tissue section placement site. It is a figure explaining the fixation process which fixes an extraction specimen tissue in preparation of a tissue microarray. It is a figure explaining the embedding process of paraffin embedding in preparation of a tissue microarray. It is a figure explaining the slice process which obtains a sliced sample by slicing in preparation of a tissue microarray. It is a figure explaining the extension process which floats and extends to 40 degreeC-50 degreeC warm water in preparation of a tissue microarray. It is a figure explaining the cutting process which scrapes a thin slice sample on the plate made from the synthetic resin which has elasticity in preparation of a tissue microarray. It is a figure explaining the cutting process which cuts in preparation of a tissue microarray, and obtains a small slice tissue section. It is a perspective external view of the porous sheet | seat for tissue dyeing | staining used in the case of incubation with an antigen activation process, a staining marker (primary antibody), and a secondary antibody. FIG. 2 is a perspective external view of a tissue microarray in which positive controls are arranged in addition to small sliced tissue sections. FIG. 3 is a perspective external view of a tissue microarray in which positive controls, negative controls, and normal tissues are arranged in addition to small sliced tissue sections. It is a photograph figure which shows the slide glass in which 21 types of dyeing marker names were described. It is a photograph figure which shows the state which piled up the porous sheet | seat for tissue dyeing | staining of silicon | silicone on a slide glass. It is a photograph figure which shows the result of the adhesive test of the porous sheet | seat for tissue dyeing | staining, and a slide glass. It is a photograph figure which shows the state which carried out the immuno-staining of 21 small slice tissue sections and a positive control with 21 types of staining markers using the porous sheet | seat for 21 tissue staining. It is a 40 times magnified photograph figure which shows the positive control of bcl-2 immunostaining and bcl-2 immunostaining of the target small slice tissue section. It is a photograph figure which shows the immuno-staining using the tissue microarray by which many small sliced tissue sections are arrange | positioned, and the porous sheet | seat for tissue staining mounted on it.

(Embodiment 1)
Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

  FIG. 1 is a perspective external view of a tissue microarray 900 according to this embodiment. As shown in FIG. 1, the tissue microarray 900 includes a plurality of small sliced tissue sections 100 cut from the same tissue of the same case of the same person on a slide plate 200.

  A conventional tissue microarray is one in which a large number of tissues of different cases of different patients are arranged on a slide plate, and these tissues are stained with a single staining marker. The tissue microarray according to the present embodiment Reference numeral 900 denotes a tissue microarray having a novel structure in which a plurality of cut thin sliced tissue sections 100 of the same tissue of the same person of the same person are arranged on the slide plate 200. The tissue analysis method of the present invention will be described later. Thus, it is a novel tissue analysis method for staining the cut sliced tissue sections 100 with a plurality of staining markers.

  Each cut small sliced tissue section 100 on the slide plate 200 is a formalin-fixed and paraffin-embedded tissue. The shape of each cut thin sliced tissue section 100 is not particularly limited, but is, for example, a substantially circular spot. When the shape of each cut small sliced tissue section 100 is circular, the size of each spot is not particularly limited, but is, for example, 0.2 mm to 10.0 mm in diameter. 2.0 mm.

  In addition, the number of each spot is set in consideration of various matters selected from those skilled in the art, such as the tissue to be examined, the case to be examined, the cancer progression stage, and the precision of the examination, and is particularly limited. However, it is 2 to 2000, for example. In the tissue microarray 900 as an example shown in FIG. 1, 21 pieces of cut sliced tissue sections 100 are arranged.

  FIG. 2 is a perspective external view of the slide plate 200 on which the cut small sliced tissue sections 100 are arranged. As shown in FIG. 2, the slide plate 200 describes a tissue section placement site 101 where the small sliced tissue section 100 is placed, and a staining marker name where the small sliced tissue section 100 is stained. The shape of the tissue section placement site 101 is, for example, a substantially circular shape. The name of the staining marker is described in the upper part of the tissue section placement site 101. The slide plate 200 is made of glass or plastic. By forming the slide plate 200 from plastic, the tissue microarray 900 can be provided at a lower cost than that of a glass carrier. As the plastic material, a thermoplastic resin can be used from the viewpoint of easy surface treatment and mass productivity. In addition, the name of the staining marker described in FIGS. 1 and 2 and the following FIG. 3 is a specific example.

  In addition, the name of the antigen activation solution suitable for the staining marker to be stained on the cut sliced tissue section 100 can be written on the slide plate 200. When tissue sections fixed with formalin or the like are immunostained, antigen-antibody reaction may not occur or may be attenuated by masking such as formaldehyde cross-linking. For this reason, an antigen activation treatment is an operation for allowing an antibody to recognize an antigen by unmasking with a heat treatment or the like. Different staining markers may result in different antigen activation solutions. However, by describing the name of the antigen activation liquid on the slide plate 200, it is possible to appropriately perform the antigen activation before the incubation with the primary antibody without making a mistake in the antigen activation liquid.

  In addition, the name of the secondary antibody suitable for the primary antibody of the staining marker to be stained on the cut sliced tissue section 100 can be described on the slide plate 200. If the secondary antibody changes due to the change in the type of the primary antibody, the name of the secondary antibody can be described on the slide plate 200 so that the secondary antibody can be appropriately operated without making a mistake. is there.

  The method of describing the name of the staining marker on the slide plate 200 is not particularly limited, but can be described (printed) with a water-soluble pigment having water resistance and solvent resistance after drying, for example. It is also possible to write the name of the staining marker on the slide plate 200 by a laser marking with a carbon dioxide laser or the like. Similarly, the tissue section placement site 101, the name of the antigen activation solution, and the name of the secondary antibody can be described (printed) with a water-soluble pigment or laser marking.

  Then, a plurality of cut thin sliced tissue sections 100 are arranged on the tissue section arrangement site 101, whereby the tissue microarray 900 shown in FIG. 1 is formed. As shown in FIG. 3, the name of the staining marker can be described inside the tissue section placement site 101. In such a case, the name of the staining marker is somewhat difficult to see by placing the cut sliced tissue section 100 in the tissue section placement site 101, but which staining marker is used for which tissue slice placement site 101 is directly used. Since it is described, the staining marker can be injected into an appropriate position without making a mistake.

  Next, a process for creating the tissue microarray 900 will be described. FIG. 4A to FIG. 4F are explanatory diagrams of the process of creating the tissue microarray 900. FIG. FIG. 4A is a diagram for explaining a fixing process for fixing an extracted specimen tissue, FIG. 4B is a diagram for explaining an embedding process for embedding paraffin, and FIG. 4C is a slice to obtain a sliced sample. FIG. 4D is a diagram for explaining an extension process in which the film is floated and stretched in warm water of about 40 ° C. to 50 ° C., and FIG. 4E is an illustration on an elastic synthetic resin plate. FIG. 4F is a diagram for explaining a cutting step for cutting and obtaining a small sliced tissue section.

  The preparation process of the tissue microarray 900 includes (A) a fixing process for fixing the extracted specimen tissue, (B) an embedding process for dehydrating the fixed extracted specimen tissue and embedding in paraffin, and (C) slicing the embedded sample. A thin-cutting process for obtaining a thin-cut sample, (D) an extension process for floating and extending the thin-cut sample in hot water of about 40 ° C. to 50 ° C., (E) A scraping step of scooping on the plate, (F) a cutting step of cutting a sliced sample on the plate with a cutting tool to obtain a sliced tissue section, and (G) a sliced sliced tissue section. Is arranged on the preparation.

  First, in the fixing step, as shown in FIG. 4A, the extracted specimen tissue 300 is fixed. That is, a chemical treatment is performed to protect the extracted specimen tissue 300 from deterioration due to autolysis and denaturation. Although the reagent used for fixation is not particularly limited, for example, formaldehyde, glutaraldehyde, or the like can be used as appropriate. The extracted sample tissue 300 is a sample tissue extracted from a living body, and is not particularly limited, but is a cancer tissue such as liver cancer, colon cancer, stomach cancer or the like.

  In the embedding process, the fixing solution adhering to the extracted specimen tissue 300 is washed with water in order to sufficiently wash it, and then dehydrated with alcohol to completely remove the water. The alcohol is gradually increased in concentration from a low concentration to be transferred to absolute alcohol in order to completely remove moisture. Next, in order to infiltrate the tissue with paraffin that does not dissolve in alcohol, a mediator such as xylol or chloroform is used, and further alcohol is removed. Then, as shown in FIG. 4B, the fixed excised specimen tissue 300 is placed in a melted liquid paraffin and sufficiently immersed, and then gradually cooled and embedded in paraffin. The shape of the ball fin block is not particularly limited, but is a prismatic shape, for example, as shown in FIG. 4B.

  Next, in the slicing step, as shown in FIG. 4C, a plurality of sliced samples 310 are obtained by slicing the extracted specimen tissue 300 embedded in paraffin. The means for slicing is not particularly limited. For example, a known slicing means such as a microtome can be used. When a microtome is used, the thickness is reduced from the order of micrometers to several tens of nanometers. Homogeneous cutting with can be performed reliably. The shape of the sliced sample 310 is not particularly limited. For example, when the shape of the paraffin block is a prismatic shape, it is a rectangle as shown in FIG. 4C. The sliced sample 310 shows a cutting site of the small sliced tissue section 100 obtained in the cutting process described later.

  Here, the tissue microarray 900 is configured to arrange a plurality of small sliced tissue sections of the same tissue of the same person on the slide plate 200. The “same tissue” is the same as shown in FIG. 4C. In the block, the x-coordinate and y-coordinate are tissues of the same part.

  Next, in the extending step, as shown in FIG. 4D, the sliced sample 310 obtained in the slicing step is floated and stretched in warm water of about 40 ° C. to 50 ° C. The aqueous solution is not particularly limited as long as it does not damage the sample. For example, water, physiological saline, and general buffers used in the field of handling biological samples can be used. In this extension process, the thin sliced sample 310 is floated and stretched in warm water of about 40 ° C. to 50 ° C. to remove wrinkles of the thin sliced sample 310 and to adhere the small sliced tissue section 100 to the slide glass. Can be increased.

  Next, in the scraping step, as shown in FIG. 4E, the sliced sample 310 floated in the aqueous solution is scraped onto a plate 320 made of synthetic resin having elasticity. Here, the elastic synthetic resin plate 320 is a plate made of a synthetic resin that is not a hard material such as glass, and examples thereof include a plate made of a material used for a cutting mat or the like. Specifically, the plate is made of a material such as an acrylic resin, a foamable fluororesin, soft vinyl chloride or vinyl chloride, a soft olefin resin such as polypropylene, or a hard olefin resin.

  Next, in the cutting step, as shown in FIG. 4F, the thin sliced sample 310 on the synthetic resin plate 320 is cut with a cutting tool to obtain a small sliced tissue section 100. By cutting the sliced sample 310 on the elastic synthetic resin plate 320, the sliced sample 310 can be cut without being damaged. The cutting tool is not particularly limited as long as it is a tool that can cut the thin cut sample 310. For example, a cylindrical cutter for collecting a skin sample, a normal cutting knife, or the like can be used.

  Finally, in the arranging step, the cut sliced tissue slice 100 is appropriately placed on the slide plate 200 at a desired position, and the desired sliced sliced tissue slice 100 as shown in FIG. 1 is combined. A tissue microarray 900 is created.

  Next, a tissue analysis method using the tissue microarray 900 created in this way will be described.

  The tissue microarray 900 is performed by immunohistochemical staining or in-situ hybridization in the same manner as a normal tissue slide specimen. In immunohistochemical staining, a specific antibody against an object (antigen) on a tissue section as a subject is reacted, and further, an antigen-antibody reaction or a chemical reaction is combined to form an immune complex of the specific antibody and a labeling enzyme, The presence of an antigen is visualized by a color reaction of a labeling enzyme. In situ hybridization uses a labeled DNA probe or RNA probe to form a hybrid with a target gene (mRNA) in a subject tissue, stains the subject cell or tissue, and the gene (mRNA) cell or tissue. It is intended to visualize the localization at.

  First, prior to staining, paraffin is removed using xylene or the like from the sliced tissue slice 100 that has been embedded in paraffin and then cut.

  Next, each cut sliced tissue section 100 is stained using a plurality of types of staining markers. FIG. 5 is a perspective external view of a tissue staining porous sheet 400 used in incubation with an antigen activation treatment, a staining marker (primary antibody), and a secondary antibody. As shown in FIG. 5, the tissue staining porous sheet 400 has a plurality of through-holes 410 formed in a base material 420, and these through-holes 410 are a plurality of cut small plates arranged in the tissue microarray 900. It is provided at a position corresponding to each of the sliced tissue sections 100. The tissue staining porous sheet 400 is formed of a transparent or translucent material and is not particularly limited, and is formed of, for example, silicon, polypropylene, polystyrene, or the like.

  In the incubation with the antigen activation treatment, the staining marker (primary antibody), and the secondary antibody, the tissue is placed on the tissue microarray 900 in a state in which each through-hole 410 and each cut sliced tissue section 100 are associated with each other. The perforated sheet for dyeing 400 is placed. Then, with the tissue staining porous sheet 400 placed on the tissue microarray 900, each of the through holes 410 of the tissue staining porous sheet 400 has an antigen activation solution and a primary antibody suitable for each of a plurality of types of staining markers. The solution and the secondary antibody solution are sequentially injected for each step and stained. For example, a transparent or semi-transparent double-sided tape is interposed between the tissue staining porous sheet 400 and the slide plate 200 to form the slide plate 200 + double-sided tape + tissue staining porous sheet 400, thereby ensuring adhesion between the two. . In addition, without interposing an adhesive layer such as a double-sided tape, the slide plate 200 and the tissue staining porous sheet 400 can be pressed against each other by physical stress to ensure adhesion between the two.

  To prevent the staining marker from drying out during the incubation, if the amount of the staining marker is too large, it may overflow to an adjacent spot. However, even if the amount of the staining marker is increased by using the tissue staining porous sheet 400 described above, since the through holes 410 form independent compartments, the staining markers accumulate in the through holes 410. , Do not overflow to the adjacent spot. In order to use the activation solution, primary antibody solution and secondary antibody solution by automation, a system can be used in which the tip of the nozzle is placed near the cut sliced tissue section 100 and pipetting is performed slowly during incubation. It is.

  Further, since the tissue staining porous sheet 400 is transparent or translucent, the name of the staining marker described on the slide plate 200 can be recognized through the tissue staining porous sheet 400. The slide plate 200 describes the tissue section placement site 101 where each tissue section is placed and the name of the staining marker where each tissue section is stained. Can be injected into. Moreover, when the name of an antigen activation liquid and the name of a secondary antibody are described in the slide plate 200, those described on the slide plate 200 can be recognized through the tissue staining porous sheet 400.

  The amount of the staining marker is not particularly limited, but when the size of the spot of the cut sliced tissue section 100 is, for example, 2.0 mm in diameter, it is, for example, 10 μL to 40 μL, and in this embodiment, 20 μL. It is.

  When the staining operation using the staining marker is completed, the tissue staining porous sheet 400 may be removed from the slide plate 200. When the antigen activation solution and the secondary antibody are different depending on the marker, this is performed without removing the tissue staining porous sheet 400. By performing tissue analysis in this manner, a comprehensive protein expression profile of each individual patient can be easily obtained, and personalized medicine can be performed at the clinical site. Therefore, the tissue microarray 900 according to the present invention is referred to as an individualized medical tissue microarray (TSMA).

(Embodiment 2)
The tissue microarray 900 of the present invention is not limited to the above-described embodiments. FIG. 6 is a perspective external view showing a tissue microarray in which a separate positive control 110 is arranged for each marker in addition to a plurality of cut sliced tissue sections 100. As shown in FIG. 6, in the tissue microarray 900 according to the second embodiment, the cut sliced tissue section 100 and the positive control 110 are disposed adjacent to each other in each tissue section placement site 101. .

  In the tissue microarray 900 according to the second embodiment, since the positive control 110 is arranged on the slide plate 200 in addition to the plurality of cut thin sliced tissue sections 100 described above, the same conditions as the positive control 110 are used. The inspection result of each of the small sliced tissue sections 100 to be inspected can be verified, and an accurate profile can be obtained.

  Moreover, as shown in FIG. 7, in addition to the cut sliced tissue section 100, a positive control 110, a negative control 120, and a normal tissue 130 are arranged adjacent to each other in each tissue section placement site 101. It is also possible. The normal tissue 130 is a normal tissue of the same case with respect to the target tissue, but when it is difficult to obtain a normal tissue of the same case, it is possible to place another person's normal tissue (a normal tissue of another case). Since the positive control 110, the negative control 120, and the normal tissue 130 are arranged on the slide plate 200 in addition to the plurality of cut sliced tissue sections 100, the positive control 110, the negative control 120, and the normal tissue 130 Under exactly the same conditions, the inspection result of each small slice tissue section 100 to be inspected can be verified, and a very accurate profile can be obtained.

<Slide glass>
As the slide plate, a MAS-coated slide glass (Matsunami Glass Industrial Co., Ltd.) having a length of 76.2 mm, a width of 25.4 mm, and a thickness of 1.0 mm was used. PX-G ink (Epson) was used as a water-soluble pigment, and circular tissue section placement sites were directly described (printed) on a slide glass in a total of 21 pieces of 3 × 7 in total. In addition, the names of the staining markers were directly described (printed) at 21 positions above the tissue section placement site described on the slide glass. The names of stained markers described on the slide glass are EstrogenReceptor (ER), Progesterone Receptor (PgR), HER2 receptor (HER2), Epithelial Growth Factor Receptor (EGFR), MIB-1, Cytokeratin5 / 6 (CK5 / 6), synaptophysin, There were 21 types: chromograninA, CD56, AndrogeneReceptor (AR), p53, p21, BRCA1, cyclinD1, E-cadherin, Smad4, Rb, c-Myc, β-catenin, ALK1, and bcl-2. In this way, a slide glass as shown in FIG. 8 was prepared.

<Tissue microarray>
Breast cancer tissue as an excised specimen tissue was fixed using formaldehyde. The fixed breast cancer tissue was thoroughly washed with water, and then gradually increased in concentration from low-concentration alcohol and then transferred to absolute alcohol, and was sufficiently dehydrated. Next, the alcohol was removed using xylol, and the fixed breast cancer tissue was placed in a melted liquid paraffin and sufficiently immersed, and then gradually cooled and embedded in paraffin. Next, a plurality of sliced samples of the same tissue of the same case of the same person were obtained by slicing the paraffin-embedded breast cancer tissue with a microtome. The thickness of the sliced sample sliced with a microtome was 4 μm. Next, these thin sliced samples were floated and stretched in warm water of about 40 ° C to 50 ° C. Next, a sliced sample floated on warm water of about 40 ° C. to 50 ° C. was scraped on a vinyl chloride cutting mat. Next, the thin slice sample scooped up on the cutting mat was cut with a cylindrical cutter, and 21 small slice tissue sections of the same tissue of the same case of the same person were obtained. Finally, the small sliced tissue section was placed in the tissue section placement site on the glass slide described above, and further, the sliced tissue section obtained from each positive control tissue in the same manner as described above at the tissue section placement site. To create a tissue microarray.

<Perforated sheet for tissue staining>
The porous sheet for tissue staining used in the incubation has almost the same shape and size as the slide glass, and 3 × 21 in the position corresponding to the tissue section placement site described on the slide glass. Through-holes were formed. FIG. 9 is a photograph showing a state in which a silicon-based porous sheet for tissue staining is placed on a slide glass. As shown in FIG. 9, the tissue staining porous sheet is a transparent material formed of silicon, and each staining marker name described on the slide glass can be recognized through the tissue staining porous sheet. It was. Moreover, as shown in FIG. 9, 21 tissue section arrangement | positioning sites described on the slide glass and 21 through-holes of the porous sheet | seat for tissue dyeing | stains corresponded completely.

  An orange transparent double-sided tape was interposed between the tissue staining porous sheet and the slide glass to test the adhesion between them. Among the 21 through holes of the tissue staining porous sheet, 11 through holes are filled with indigo carmine as a coloring pigment, and the remaining 10 through holes are filled with PBS. Incubation was performed at 37 ° C. for 40 minutes.

  FIG. 10 is a photograph showing the results of an adhesion test between the tissue staining porous sheet and the slide glass. The dark blue liquid is indigo carmine and the clear liquid is PBS. As shown in FIG. 10, after the incubation, indigo carmine and PBS were stored in the through hole of the tissue staining porous sheet without leaking. In addition, it did not leak even in incubation at 95 ° C. for 40 minutes, and it did not leak even in over night.

<Immunostaining>
Next, using the above tissue staining porous sheet, it was placed on the tissue microarray prepared in the above <tissue microarray>, and immunostaining was performed using 21 types of staining markers as shown below. Went. That is, antigen activation at 121 ° C. for 15 minutes at tris-EDTA pH 10 was followed by hydrogen peroxide methanol treatment for 15 minutes and blocking with 10% goat serum for 5 minutes. A 21-hole porous sheet for tissue staining with one side of a double-sided tape attached was placed on and adhered to the tissue microarray, and the through-holes were filled with PBS. Thereafter, PBS was removed one by one, and 20 μl of the primary antibody identical to the described staining marker name was injected. The staining markers used were EstrogenReceptor (ER), Progesterone Receptor (PgR), HER2 receptor (HER2), Epithelial GrowthFactor Receptor (EGFR), MIB-1, Cytokeratin5 / 6 (CK5 / 6), synaptophysin, chromograninA, CD56, Androgene Receptor (AR), p53, p21, BRCA1, cyclinD1, E-cadherin, Smad4, Rb, c-Myc, β-catenin, ALK1, and bcl-2.

  After incubation at 37 ° C. for 40 minutes, the plate was rinsed with PBS, and the tissue staining porous sheet was removed. In addition, the secondary antibody was incubated for 10 minutes. Next, after rinsing with PBS, treatment with an HRP streptavidin solution was performed for 10 minutes, followed by rinsing with PBS and color development with DAB. After counterstaining with hematoxylin, it was sealed and encapsulated.

  FIG. 11 is a photographic diagram showing a state in which 21 tissue sections and positive controls were immunostained with 21 types of staining markers using a 21-hole porous sheet for tissue staining. In FIG. 11, a dark brown tissue piece located on the right side is a positive control, and a tissue piece located on the left side is a small sliced tissue section as a target. In addition, the described tissue section placement site and staining marker name were not peeled off in all steps of immunostaining, such as deparaffinization and antigen activation with a tris-EDTA solution at 121 ° C. for 15 minutes. As shown in FIG. 11, in the tissue microarray of the present invention, a plurality of small sliced tissue sections of the same tissue of the same person of the same person are arranged on a slide plate, and these small sliced tissue sections are a plurality of stained markers. Since each is stained at the same time, a comprehensive protein expression profile of each individual patient can be acquired, and personalized medicine becomes possible.

  FIG. 12 is a 40-fold enlarged photograph showing a positive control of bcl-2 immunostaining and bcl-2 immunostaining of a target thin sliced tissue section. In FIG. 12, the staining on the right side is a positive control of bcl-2 immunostaining, and the staining on the left side is bcl-2 immunostaining of a small slice of target tissue. As shown in FIG. 12, the target small slice tissue section and the positive control are adjacent to each other, and both are stained under the same conditions. Therefore, bcl-2 negative determination of the left target tissue (breast cancer tissue) is extremely accurate and easy.

  FIG. 13 is another example, and is a photographic diagram showing immunostaining using a tissue microarray in which more small sliced tissue sections are arranged and a tissue staining porous sheet placed thereon. is there. As shown in FIG. 13, 34 small sliced tissue sections are arranged in the tissue microarray, and 36 through-holes of 4 × 9 are formed on the tissue microarray. A perforated sheet was placed. Then, immunostaining was performed using 34 types of staining markers.

  As shown in FIG. 13, a more detailed comprehensive protein expression profile can be obtained by arranging a large number of small sliced tissue sections of the same tissue of the same case of the same person on the slide plate, further promoting personalized medicine It becomes possible to make it.

  The tissue microarray according to the present invention is used to obtain a comprehensive protein expression profile of an individual in the clinical field and in fields such as medical and basic medical research, and makes a great contribution to personalized medicine especially in the clinical field. .

100: Small slice tissue section 101: Tissue section placement site 110: Positive control 120: Negative control 130: Normal tissue 200: Slide plate 300: Extracted specimen tissue 310: Slice specimen 320: Plate 400: Porous sheet for tissue staining 410 : Through hole 420: Base material 900: Tissue microarray

Claims (11)

  A tissue microarray, wherein a plurality of small sliced tissue sections of the same tissue of the same person and the same case are arranged on a slide plate.   The tissue microarray according to claim 1, wherein, in addition to a plurality of small sliced tissue sections, a separate positive control for each marker is arranged on the slide plate.   The tissue microarray according to claim 1 or 2, wherein, in addition to a plurality of small sliced tissue sections, a separate negative control for each marker is arranged on the slide plate.   The tissue microarray according to any one of claims 1 to 3, wherein in addition to a plurality of small sliced tissue sections, normal tissues of the same case or other cases with respect to a target tissue are arranged on the slide plate. .   The tissue microarray according to any one of claims 1 to 4, wherein a tissue section placement site indicating a site on which the small sliced tissue slice is placed is described on the slide plate.   The tissue microarray according to any one of claims 1 to 5, wherein a name of a staining marker for staining the small sliced tissue section is described on the slide plate.   The tissue according to any one of claims 1 to 6, wherein a name of an antigen activation solution suitable for a staining marker for staining the sliced tissue section is described on the slide plate. Microarray.   8. The name of a secondary antibody suitable for a primary antibody of a staining marker for staining the sliced tissue section is described on the slide plate. Tissue microarray.   9. The description of the tissue section placement site, the staining marker name, the antigen activation solution, or the name of the secondary antibody is written on the slide plate by laser engraving. The tissue microarray according to claim 1.   A tissue analysis method comprising arranging a plurality of small sliced tissue sections of the same tissue of the same person in the same case on a slide plate, and staining each of the small sliced tissue sections with a plurality of staining markers.   On the slide plate, a tissue staining sheet having a plurality of through-holes provided in a base material is placed, and an antigen activation solution suitable for each staining marker and a plurality of staining are disposed in the plurality of through-holes of the tissue staining sheet. The tissue analysis method according to claim 10, wherein the marker and a secondary antibody suitable for the primary antibody of each staining marker are each injected and stained.