CN113588375A - High-stability bone and bone tumor tissue microarray chip and preparation method thereof - Google Patents
High-stability bone and bone tumor tissue microarray chip and preparation method thereof Download PDFInfo
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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Abstract
The invention relates to a high-stability bone and bone tumor tissue microarray chip and a preparation method thereof, and the bone and bone tumor tissue microarray chip prepared by the method better solves the problems of chip dropping, few effective target tissues, cell degeneration and the like.
Description
Technical Field
The invention belongs to the technical field of tissue chips, and particularly relates to a high-stability bone and bone tumor tissue microarray chip and a preparation method thereof.
Background
Tissue chips, also called tissue microarrays, are an important branch of biochip technology, where many different individual tissue specimens are arranged on the same carrier (most slides are used) in a regular array, mainly for studying the expression of the same gene or protein molecule in different cells or tissues. Since the emergence of 1998, the technology is widely popularized and applied due to the advantages of large scale, high flux, standardization and the like. The method has the greatest advantages that the experimental conditions of the tissue samples on the chip are completely consistent, and the quality control is excellent. The time and reagent saving are more obvious.
The tissue chip preparation technology in China is mature day by day, but for some special tissues such as bone tissues and bone tumor tissues, because the hardness is high, the acidity after decalcification is strong, and the tissue adhesion is poor, the preparation of the high-quality tissue chip is difficult, the preparation is time-consuming and labor-consuming, the phenomena of point dropping and piece dropping frequently occur, and the subsequent experimental results are directly influenced by the poor quality of the tissue chip. In addition, the bone tumor tissue chip has a large content of effective target tissue cells due to the influence of tissue decalcification and the like, and the number and the quality of the effective target tissue in the tissue points of the tissue chip cannot be guaranteed because the cells are denatured by unstable factors of early tissue treatment such as fixation, decalcification and the like.
The present invention is particularly highlighted in view of the above reasons.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a high-stability bone and bone tumor tissue microarray chip and a preparation method thereof.
The first purpose of the invention is to provide a preparation method of a microarray chip for high-stability bone and bone tumor tissues, which comprises the following steps:
(1) collecting bone and bone tumor tissue samples, and processing the tissue samples;
(2) embedding the processed tissue sample in paraffin, then carrying out section HE staining, and scanning an HE staining piece;
(3) the preparation method comprises the steps of diagnosing under a microscope by a pathologist in combination with a scanning image, marking effective target tissues, manufacturing a tissue chip receptor wax block according to a designed tissue microarray and the size of a point, contrasting an effective target tissue area marked by a HE section, punching and sampling a donor wax block, taking out a sample column core, embedding the sample column core into a receptor wax block hole, after all embedding is finished, placing the tissue microarray wax block in a thermostat at 57 ℃ for 1.5h to fix wax and fuse, then placing the tissue microarray wax block at room temperature for 4h, transferring the tissue microarray wax block to a refrigerator for preservation at 2-6 ℃ for more than 6 h, and carrying out slicing, baking and HE conventional dyeing to prepare the high-stability bone and bone tumor tissue microarray chip.
The wax block of the invention is prepared from lycra wax with a melting point of 52-56 ℃.
Further, the tissue sample treatment in step (1) specifically comprises: fixing the bone and bone tumor samples in 10% formalin solution for 24 hr, sawing bone slices or bone tumor tissues of 0.5cm thickness, fixing in 10% formalin solution for 48 hr, placing the bone or bone tumor tissues in decalcifying agent until the bone tissues are easy to be penetrated by needle, dewatering, transparentizing, waxing and slicing.
The decalcifying agent is prepared by adding hydrochloric acid decalcifying liquid, concentrated hydrochloric acid 15ml, sodium chloride 7.5ml and distilled water to 100 ml. The decalcifying agent needs to be replaced every day until the bone tissue is easily penetrated by the needle, washed with running water for 24 hours to remove the acid, and then processed for the next step.
Further, in the step (3), the tissue microarray wax block is placed at 54 ℃ for 1 hour to solidify and fuse the wax, then is placed at room temperature for 4 hours, and is refrigerated and stored at 4 ℃ for more than 6 hours.
The inventor finds that the tissue column core is fully compatible with the receptor wax block and avoids dropping of the spot sheet by adopting the temperature and time for wax fixation, room-temperature placement and specific-temperature refrigeration.
The wax dipping process of the invention is as follows:
(1) 75% alcohol, 37 degrees, 2 hours; (2) 95% alcohol, 37 degrees, 2 hours; (3) 95% alcohol, 37 degrees, 2 hours; (4) 100% alcohol, 37 degrees, 2 hours; (5) 100% alcohol, 37 degree, 1.5-2 hours; (6) xylene I, 45 minutes; (7) xylene II, 45 minutes; (8) paraffin I, 60 ℃ for 30 minutes; (9) paraffin II, 60 ℃ for 60 minutes; (10) paraffin III, 60 degrees, 90 minutes.
The embedding operation in the invention is as follows:
the main switch of the power supply of the embedding machine is turned on two hours in advance, the embedding machine is in a working state, the temperature is increased, and the temperature of the embedding wax is increased to about 58 ℃. The freezing table was opened before embedding. Taking out the embedding model, controlling the wax amount by pedaling, clamping a tissue block after injecting a small amount of paraffin, completely flattening the tissue, specifically setting the embedding direction according to the tissue shape and anatomical structure, such as erecting tubular and skin tissues for embedding, then pressing an embedding box (5mm in thickness), injecting a small amount of paraffin on the embedding box, placing the embedding box on a freezing table after embedding, taking out the wax block when the surface of the embedding block on the freezing table is coagulated, trimming peripheral residual wax, arranging the wax blocks in sequence, and placing a freezing chamber of a refrigerator (18 ℃ below zero) for later use.
The HE staining of the invention is specifically performed as follows:
fixing the embedded wax block on a slicer, cutting into thin wax sheets with the thickness of 3 microns, folding and superposing the thin wax sheets which are continuously cut, obliquely pulling the thin wax sheets at an angle of 25-30 degrees into heated water (the water temperature is 45 ℃) to be ironed, sticking the thin wax sheets onto a glass slide, and drying the glass slide for one hour in a 45 ℃ thermostat.
(1) The dried slide glass with the thin tissue piece is inserted into a staining rack in sequence and softly; (2) xylene I, 5 minutes; (3) xylene II, 10 minutes; (4) absolute ethyl alcohol I, 1 minute; (5) absolute ethyl alcohol II, 1 minute; (6) 95% alcohol I, 1 minute; (7) 95% alcohol II, 1 minute; (8) 90% alcohol, 1 minute; (9) 85% alcohol, 1 minute; (10) staining with hematoxylin for 1 minute; (11) washing with tap water for 1 minute; (12) differentiating with 1% hydrochloric acid alcohol for 20 s; (13) washing with tap water for 1 minute; (14) 1% ammonia water is anti-blue for 30 seconds; (15) washing with distilled water for 1 minute; (16) eosin staining for 1 min; (17) washing with tap water for 1 minute; (18) 1 minute with 85% alcohol; (19) 90% alcohol for 1 minute; (20) 1 minute with 95% alcohol; (21) anhydrous ethanol I, 1 minute and 30 seconds; (22) anhydrous ethanol II, 2 min 30 s; (23) xylene I, 1 minute; (24) xylene II, 1 minute; (25) sealing neutral gum into a sheet; (26) and (3) dyeing results: the cell nuclei were blue and the cytoplasm was pink.
The scanning (40 times multiplying power) of the HE (hematoxylin-eosin staining) staining slice specifically comprises the following steps:
(1) labeling the left-hand frosted area of the glass slide, wherein the contents comprise Zhongke Guanghua logo, serial number and HE character;
(2) sequentially putting the scanning slide disc into a scanner and slightly pushing the scanning slide disc into the scanner (a scanning instrument of a model 500B of a superior PRECICE);
(3) automatically identifying images and focusing for scanning;
(4) the scanned cells are clear and bright in color when the scanned image is opened for observation.
The second purpose of the invention is to provide a high-stability bone and bone tumor tissue microarray chip prepared by the method.
In the present invention, a Beecher MTA-1 array apparatus was used.
Furthermore, the high-stability bone and bone tumor tissue microarray chip comprises a glass slide substrate, a special label and a sample application tissue with target information, wherein the sample application tissue is attached to the glass slide.
Furthermore, the high-stability bone and bone tumor tissue microarray chips are arranged in a single bone tumor matrix or in a multi-pathological bone tumor matrix.
Furthermore, the single bone tumor matrix is arranged into a single bone tumor matching and/or non-matching paraneoplastic, tumor margin and normal bone tissue combination matrix, and the multi-pathological bone tumor matrix is arranged into a multi-pathological bone tumor matching and/or non-matching paraneoplastic, tumor margin and normal bone tissue combination matrix.
Furthermore, the paraneoplastic, tumor margin and normal bone tissue are one point or a plurality of points.
Can be completely matched (all bone tumor tissue points on the chip are matched with paraneoplastic and/or paraneoplastic marginal tissues of the same example) or incompletely matched (all bone tumors on the chip are matched with paraneoplastic and marginal tissues of the same example and/or part of different examples). The normal bone tissue can be matched or not matched, and can be placed at any point of the tissue microarray (including a marking point).
Furthermore, the multiple tissue points containing bone mass are positioned in the center or near the center area of the tissue microarray chip, and tissues with less bone mass and abundant tumor cells are placed close to the tissue points on the sides and the rows.
Furthermore, the tissue microarray chip is used for bone tumor immunohistochemistry, nucleic acid in situ hybridization, fluorescent nucleic acid in situ hybridization or in situ PCR detection.
Compared with the prior art, the invention has the beneficial effects that:
the bone and bone tumor tissue microarray chip prepared by the method better solves the problems of chip dropping, few effective target tissues, cell degeneration and the like, comprehensively designs the optimal bone tumor tissue chip arrangement mode from the aspects of physics, plane arrangement and practicability, saves time and cost, improves the core quality of the tissue chip, obtains extensive scientific research in the fields of antibody research, tumor research, gene detection and the like, is favorable for clinical and teaching institutions, and basically meets the requirements of research and development of targeted drugs, tumor diagnosis, generation mechanisms and returning of the targeted drugs on tumor biotherapy in the fields of tissues, proteins, gene molecules and the like, thereby improving the scientific research level more quickly, conveniently, comprehensively and strictly.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a drawing of a bone and bone marrow tissue chip according to the present invention including 10 microarray chips of normal bone and bone marrow tissues;
FIG. 2 is an arrangement of a first microarray chip of the present invention;
FIG. 3 is a diagram of a microarray chip for osteosarcoma and normal bone tissue of the present invention comprising 50 cases of osteosarcoma and 1 case of normal bone tissue;
FIG. 4 is an arrangement of a second microarray chip of the present invention;
FIG. 5 is a diagram of microarray chips containing 70 cases of osteosarcoma and 1 case of normal bone tissue according to the present invention;
FIG. 6 is an arrangement of a third microarray chip of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The glass slide in the present invention may be glass such as: the anti-falling/adhesion immune combined glass slide, immunofluorescence in situ hybridization glass slide, polylysine glass slide, or silicification anti-falling glass slide and biological membrane.
The slide glass substrate of the invention is provided with target information sample application tissues, and the diameter of each point can be 0.6mm, 1.0mm, 1.5mm and 2.0mm according to different requirements. The tissue core spacing and the tissue core diameter are as follows: the 3 ratios are respectively 0.2mm, 0.3mm, 0.5mm and 0.6 mm.
From left to right, arranged in rows and columns, each row in the horizontal direction numbered with a number, and each column in the vertical direction numbered with a letter, starting from 1-A, respectively. Each slide base may vary from 702 to 4 points. The last point of the labeled microarray is the labeled point (which may be bone or bone tumor tissue, or carbon core, etc.), and the end of the labeled microarray is shown in FIG. 1. The arrangement mode can be a rectangular matrix array mode, a square matrix array mode and other shape arrangements as shown in fig. 2-6, wherein the tissue of the italic point in fig. 4 and 6 has less bone content and abundant tumor cells.
The invention can also make the organization chip of the permutation of the way of the array of the multicomponent microarray according to the need, namely divide the area of the symmetrical or asymmetric matrix array in the recipient block of the array, can be the rectangle, can be the square matrix array, according to studying the design model, increase the row spacing and/or column spacing and divide a plurality of matrix array combinations. The row or column spacing may be 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.8mm, 1.0mm, 1.5mm, 2.0mm, 3.0 mm. The slide glass can also be fished continuously or discontinuously (one or more slices are separated) when the slide glass is sliced according to the research requirement, and the slide glass is symmetrically mounted on the slide glass at the interval of 1 tissue core diameter or 2 tissue core diameter distances.
The wax impregnation process described in the following examples is specifically as follows:
(1) 75% alcohol, 37 degrees, 2 hours; (2) 95% alcohol, 37 degrees, 2 hours; (3) 95% alcohol, 37 degrees, 2 hours; (4) 100% alcohol, 37 degrees, 2 hours; (5) 100% alcohol, 37 degree, 1.5-2 hours; (6) xylene I, 45 minutes; (7) xylene II, 45 minutes; (8) paraffin I, 60 ℃ for 30 minutes; (9) paraffin II, 60 ℃ for 60 minutes; (10) paraffin III, 60 degrees, 90 minutes.
The embedding operation is as follows:
the main switch of the power supply of the embedding machine is turned on two hours in advance, the embedding machine is in a working state, the temperature is increased, and the temperature of the embedding wax is increased to about 58 ℃. The freezing table was opened before embedding. Taking out the embedding model, controlling the wax amount by pedaling, clamping a tissue block after injecting a small amount of paraffin, completely flattening the tissue, specifically setting the embedding direction according to the tissue shape and anatomical structure, such as erecting tubular and skin tissues for embedding, then pressing an embedding box (5mm in thickness), injecting a small amount of paraffin on the embedding box, placing the embedding box on a freezing table after embedding, taking out the wax block when the surface of the embedding block on the freezing table is coagulated, trimming peripheral residual wax, arranging the wax blocks in sequence, and placing a freezing chamber of a refrigerator (18 ℃ below zero) for later use.
The HE staining of the invention is specifically performed as follows:
fixing the embedded wax block on a slicer, cutting into thin wax sheets with the thickness of 3 microns, folding and superposing the thin wax sheets which are continuously cut, obliquely pulling the thin wax sheets at an angle of 25-30 degrees into heated water (the water temperature is 45 ℃) to be ironed, sticking the thin wax sheets onto a glass slide, and drying the glass slide for one hour in a 45 ℃ thermostat.
(1) The dried glass slides with the thin tissue slices are gently kneaded and inserted into a staining rack in sequence; (2) xylene I, 5 minutes; (3) xylene II, 10 minutes; (4) absolute ethyl alcohol I, 1 minute; (5) absolute ethyl alcohol II, 1 minute; (6) 95% alcohol I, 1 minute; (7) 95% alcohol II, 1 minute; (8) 90% alcohol, 1 minute; (9) 85% alcohol, 1 minute; (10) staining with hematoxylin for 1 minute; (11) washing with tap water for 1 minute; (12) differentiating with 1% hydrochloric acid alcohol for 20 s; (13) washing with tap water for 1 minute; (14) 1% ammonia water is anti-blue for 30 seconds; (15) washing with distilled water for 1 minute; (16) eosin staining for 1 min; (17) washing with tap water for 1 minute; (18) 1 minute with 85% alcohol; (19) 90% alcohol for 1 minute; (20) 1 minute with 95% alcohol; (21) anhydrous ethanol I, 1 minute and 30 seconds; (22) anhydrous ethanol II, 2 min 30 s; (23) xylene I, 1 minute; (24) xylene II, 1 minute; (25) sealing neutral gum into a sheet; (26) and (3) dyeing results: the cell nuclei were blue and the cytoplasm was pink.
The scanning (40 times multiplying power) of the HE (hematoxylin-eosin staining) staining slice specifically comprises the following steps:
(1) labeling the left-hand frosted area of the glass slide, wherein the contents comprise Zhongke Guanghua logo, serial number and HE character;
(2) sequentially putting the scanning slide disc into a scanner and slightly pushing the scanning slide disc into the scanner (a scanning instrument of a model 500B of a superior PRECICE);
(3) automatically identifying images and focusing for scanning;
(4) the scanned cells are clear and bright in color when the scanned image is opened for observation. The second purpose of the invention is to provide a high-stability bone and bone tumor tissue microarray chip prepared by the method.
Example 1
The preparation method of the microarray chip for high-stability bone and bone tumor tissues of the embodiment comprises the following steps:
(1) collecting bone and bone tumor tissue samples, and processing the tissue samples;
the tissue sample is specifically treated as follows: fixing bone and bone tumor samples in 10% formalin solution for 24 hours, sawing bone slices or bone tumor tissues with the thickness of 0.5cm, fixing the bone and bone tumor samples in 10% formalin solution for 48 hours, placing the bone or bone tumor tissues in a decalcifying agent, adding 15ml of concentrated hydrochloric acid, 7.5ml of sodium chloride and distilled water to 100ml to obtain the decalcifying agent, replacing a new decalcifying agent every day until the bone tissues are easily punctured by a needle, dehydrating, transparentizing, soaking in wax and slicing;
(2) embedding the processed tissue sample in paraffin, then carrying out section HE staining, and scanning an HE staining piece;
(3) the preparation method comprises the steps of diagnosing under a microscope by a pathologist in combination with a scanning image, marking effective target tissues, manufacturing a tissue chip receptor wax block according to a designed tissue microarray and the size of a point, contrasting an effective target tissue area marked by a HE section, punching and sampling a donor wax block, taking out a sample column core, embedding the sample column core into a receptor wax block hole, placing the tissue microarray wax block in a thermostat at 57 ℃ for 1.5h for wax fixation and fusion after all embedding is finished, then placing the tissue microarray wax block at room temperature for 4h, transferring the tissue microarray wax block to a refrigerator at 2 ℃ for preservation for more than 6 h, and carrying out slicing, baking and HE conventional dyeing to prepare the high-stability bone and bone tumor tissue microarray chip.
The high-stability bone and bone tumor tissue microarray chip comprises a glass slide substrate, a special label and a sample application tissue with target information, wherein the sample application tissue is attached to the glass slide; the high-stability bone and bone tumor tissue microarray chip is arranged in a single bone tumor matrix; the single bone tumor matrix is arranged to match the single bone tumor; the multiple tissue points with bone content are positioned in the center or near the center area of the tissue microarray chip, and tissues with less bone content and abundant tumor cells are placed close to the side row and column tissue points.
Example 2
The preparation method of the microarray chip for high-stability bone and bone tumor tissues of the embodiment comprises the following steps:
(1) collecting bone and bone tumor tissue samples, and processing the tissue samples;
the tissue sample is specifically treated as follows: fixing bone and bone tumor samples in 10% formalin solution for 24 hours, sawing bone slices or bone tumor tissues with the thickness of 0.5cm, fixing the bone and bone tumor samples in 10% formalin solution for 48 hours, placing the bone or bone tumor tissues in a decalcifying agent, adding 15ml of concentrated hydrochloric acid, 7.5ml of sodium chloride and distilled water to 100ml to obtain the decalcifying agent, replacing a new decalcifying agent every day until the bone tissues are easily punctured by a needle, dehydrating, transparentizing, soaking in wax and slicing;
(2) embedding the processed tissue sample in paraffin, then carrying out section HE staining, and scanning an HE staining piece;
(3) the preparation method comprises the steps of diagnosing under a microscope by a pathologist in combination with a scanning image, marking effective target tissues, manufacturing a tissue chip receptor wax block according to a designed tissue microarray and the size of a point, contrasting an effective target tissue area marked by a HE section, punching and sampling a donor wax block, taking out a sample column core, embedding the sample column core into a receptor wax block hole, placing the tissue microarray wax block in a thermostat at 57 ℃ for 1.5h to fix wax and fuse after all embedding is finished, then placing the tissue microarray wax block at room temperature for 4h, transferring the tissue microarray wax block to a refrigerator at 4 ℃ for preserving for more than 6 h, and carrying out slicing, baking and HE conventional dyeing to prepare the high-stability bone and bone tumor tissue microarray chip.
The high-stability bone and bone tumor tissue microarray chip comprises a glass slide substrate, a special label and a sample application tissue with target information, wherein the sample application tissue is attached to the glass slide; the high-stability bone and bone tumor tissue microarray chip is arranged in a multi-pathological bone tumor matching and/or mismatching paraneoplastic, tumor edge and normal bone tissue combination matrix; the paraneoplastic, tumor margin and normal bone tissue are one point or one multiple points; the multiple tissue points with bone content are positioned in the center or near the center area of the tissue microarray chip, and tissues with less bone content and abundant tumor cells are placed close to the side row and column tissue points.
Example 3
The preparation method of the microarray chip for high-stability bone and bone tumor tissues of the embodiment comprises the following steps:
(1) collecting bone and bone tumor tissue samples, and processing the tissue samples;
the tissue sample is specifically treated as follows: fixing bone and bone tumor samples in 10% formalin solution for 24 hours, sawing bone slices or bone tumor tissues with the thickness of 0.5cm, fixing the bone and bone tumor samples in 10% formalin solution for 48 hours, placing the bone or bone tumor tissues in a decalcifying agent, adding 15ml of concentrated hydrochloric acid, 7.5ml of sodium chloride and distilled water to 100ml to obtain the decalcifying agent, replacing a new decalcifying agent every day until the bone tissues are easily punctured by a needle, dehydrating, transparentizing, soaking in wax and slicing;
(2) embedding the processed tissue sample in paraffin, then carrying out section HE staining, and scanning an HE staining piece;
(3) the preparation method comprises the steps of diagnosing under a microscope by a pathologist in combination with a scanning image, marking effective target tissues, manufacturing a tissue chip receptor wax block according to a designed tissue microarray and the size of a point, contrasting an effective target tissue area marked by a HE section, punching and sampling a donor wax block, taking out a sample column core, embedding the sample column core into a receptor wax block hole, placing the tissue microarray wax block in a thermostat at 57 ℃ for 1.5h to fix wax and fuse after all embedding is finished, then placing the tissue microarray wax block at room temperature for 4h, transferring the tissue microarray wax block to a refrigerator at 6 ℃ for preserving for more than 6 h, and carrying out slicing, baking and HE conventional dyeing to prepare the high-stability bone and bone tumor tissue microarray chip.
The high-stability bone and bone tumor tissue microarray chip comprises a glass slide substrate, a special label and a sample application tissue with target information, wherein the sample application tissue is attached to the glass slide; the high-stability bone and bone tumor tissue microarray chip is arranged in a single bone tumor matrix; the single bone tumor matrix is arranged to match the single bone tumor; the multiple tissue points with bone content are positioned in the center or near the center area of the tissue microarray chip, and tissues with less bone content and abundant tumor cells are placed close to the side row and column tissue points.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A preparation method of a high-stability bone and bone tumor tissue microarray chip is characterized by comprising the following steps:
(1) collecting bone and bone tumor tissue samples, and processing the tissue samples;
(2) embedding the processed tissue sample in paraffin, then carrying out section HE staining, and scanning an HE staining piece;
(3) the preparation method comprises the steps of diagnosing under a microscope by a pathologist in combination with a scanning image, marking effective target tissues, manufacturing a tissue chip receptor wax block according to a designed tissue microarray and the size of a point, contrasting an effective target tissue area marked by a HE section, punching and sampling a donor wax block, taking out a sample column core, embedding the sample column core into a receptor wax block hole, after all embedding is finished, placing the tissue microarray wax block in a thermostat at 57 ℃ for 1.5h to fix wax and fuse, then placing the tissue microarray wax block at room temperature for 4h, transferring the tissue microarray wax block to a refrigerator for preservation at 2-6 ℃ for more than 6 h, and carrying out slicing, baking and HE conventional dyeing to prepare the high-stability bone and bone tumor tissue microarray chip.
2. The method for preparing a microarray chip of highly stable bone and bone tumor tissue according to claim 1, wherein the step (1) of processing the tissue sample comprises: fixing the bone and bone tumor samples in 10% formalin solution for 24 hr, sawing bone slices or bone tumor tissues of 0.5cm thickness, fixing in 10% formalin solution for 48 hr, placing the bone or bone tumor tissues in decalcifying agent until the bone tissues are easy to be penetrated by needle, dewatering, transparentizing, waxing and slicing.
3. The method for preparing a microarray chip of highly stable bone and bone tumor tissue according to claim 1, wherein in step (3), the tissue microarray wax block is placed at 54 ℃ for 1 hour for wax fixation and fusion, then placed at room temperature for 4 hours, and then stored at 4 ℃ for more than 6 hours under refrigeration.
4. A microarray chip for highly stable bone and bone tumor tissue prepared by the method of any one of claims 1 to 3.
5. The microarray chip of claim 4, wherein the microarray chip comprises a slide substrate, a specific label and a tissue sample with target information attached to the slide substrate.
6. The microarray chip for bone tumor tissue with high stability of claim 4, wherein the microarray chip for bone tumor tissue with high stability is arranged in a single bone tumor matrix or in a multi-pathological bone tumor matrix.
7. The microarray chip for bone tumor tissue with high stability according to claim 6, wherein the single bone tumor matrix is arranged in a single bone tumor matching and/or non-matching paraneoplastic, tumor margin and normal bone tissue combined matrix, and the multi-pathological bone tumor matrix is arranged in a multi-pathological bone tumor matching and/or non-matching paraneoplastic, tumor margin and normal bone tissue combined matrix.
8. The microarray chip of claim 7, wherein the paraneoplastic, tumor margin and normal bone tissue are one or more spots.
9. The microarray chip of claim 4, wherein the multiple tissue spots with bone mass are located in the central or near-central area of the microarray chip, and the tissue spots with low bone mass and abundant tumor cells are located near the side rows and columns.
10. The microarray chip of claim 4, wherein the microarray chip is used for immunohistochemistry, in situ hybridization of nucleic acid, in situ hybridization of fluorescent nucleic acid, or in situ PCR detection of bone tumor.
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