Multi-element integrated multi-organ normal tissue microarray chip and implementation method thereof

Abstract

The invention relates to the technical field of tissue microarray chips, in particular to a multi-element integrated multi-organ normal tissue microarray chip and an implementation method thereof. The multi-element integrated multi-organ normal tissue microarray chip mainly comprises a glass slide base, a special label for Zhongsha and a sample application tissue sheet with target information, wherein the sample application tissue sheet is attached to a glass slide; a target information sample application tissue piece is carried on the glass slide base, and the tissue point spacing of the target information sample application tissue piece corresponds to the preset tissue point diameter; wherein the target information sample application tissues on the glass slide base are arranged from left to right according to a preset row and column mode, in the horizontal direction, from left to right, each row is numbered by numbers in sequence, in the vertical direction, each column is numbered according to an alphabetic sequence, and 702-4 points can not be equal on each glass slide base. The technical scheme provided by the invention has the advantages of high throughput, high efficiency, parallelism, large information amount, small error and the like.

Description

Technical Field

[0001] The present application relates to the technical field of tissue microarray chips, and in particular to a multi-element integrated multi-organ normal tissue microarray chip and a method for realizing the same. Background Art

[0002] Tissue chip technology occupies an important position in modern medical research, especially playing a key role in the fields of pathology research and drug development.

[0003] Internationally, the application scope of tissue chips has gradually expanded from the early focus on tumor molecular pathology research to all aspects of clinical pathology and physiological research. It has become a standardized routine tool, widely used in the exploration of new molecular markers in a large number of samples and the study of changes in gene expression. In the process of disease-targeted drug development, tissue chips can quickly screen out genes related to the disease, and through the analysis of large samples, it effectively speeds up the speed of drug development. At the same time, in complex analysis scenarios of multiple genes and multiple factors, tissue chips provide researchers with a powerful means to help reveal the potential mechanisms of disease occurrence and development.

[0004] When conducting protein and molecular gene detection experiments, whether for human, animal or tumor tissue chips, the corresponding normal tissue chip control is indispensable. As a control, normal tissue chips can provide a key reference standard for experimental results. Without normal tissue chip controls, the experimental results will be difficult to present in full, and the conclusions obtained will lack sufficient persuasiveness. Moreover, the rationality of the control group setting has a profound impact on the reliability of the experimental conclusions, and unreasonable control group settings cannot effectively support the experimental conclusions. In experiments involving multiple factors, it is particularly important to set up a reasonable control group. Blind settings may lead to problems such as incomplete or excessive control tissues, which will not only have an irreversible negative impact on the experimental results, but also significantly increase the cost of the experiment and consume a lot of time and energy. Therefore, when a normal control tissue chip is required for protein and molecular gene experimental research, choosing a suitable and efficient normal tissue chip as a control will have a multiplier effect on improving scientific research efficiency, scientific research level and quality.

[0005] In recent years, my country's tissue chip technology has made significant progress, and the scale of the industry has gradually taken shape. However, compared with advanced foreign technologies, there is still a certain gap, especially in the design and production of normal positive (negative) control tissue chips. For example, some positive (negative) control tissue chip designs are not targeted enough or lack universality, and the arrangement of microarray tissue chips is not practical enough in actual applications, and cannot meet the needs of large sample disease (tumor) experiments in protein and molecular research. These shortcomings have, to a certain extent, restricted the further development of my country's tissue chip technology in scientific research and clinical applications. There is an urgent need for an improved technical solution to make up for the existing defects and improve the overall level of my country's tissue chip technology to better serve medical research and clinical practice. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a multi-element integrated multi-organ normal tissue microarray chip and an implementation method thereof, so as to solve the related problems existing in the prior art.

[0007] According to a first aspect of an embodiment of the present invention, a multi-element integrated multi-organ normal tissue microarray chip is provided, wherein the multi-element integrated multi-organ normal tissue microarray chip is mainly composed of a glass slide substrate, a Zhongke Guanghua special label, and a tissue piece with target information spots attached to the glass slide;

[0008] Wherein, the slide in the slide substrate includes: a glass slide, a siliconized anti-shedding slide and a biofilm;

[0009] The target information dotted tissue slice is carried on the glass slide substrate, and the distance between tissue points of the target information dotted tissue corresponds to the preset tissue point diameter; wherein the diameter of the tissue point of the target information dotted tissue can be 0.6mm, 1.0mm, 1.5mm or 2.0mm; wherein the target information dotted tissue slice carries a preset number of target information dotted tissues;

[0010] Among them, the target information dot organization on the slide base is arranged from left to right in a preset row and column manner. From left to right in the horizontal direction, each row is numbered in sequence, and each column is numbered in alphabetical order in the vertical direction. There can be 702 to 4 dots on each base slide.

[0011] Furthermore, the chips are arranged in a rectangular matrix, a square matrix or other shapes, and normal tissue control chips can be arranged in a variety of combined microarrays according to preset requirements;

[0012] Among them, the row or column spacing can be 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.8mm, 1.0mm, 1.5mm, 2.0mm, 3.0mm;

[0013] Alternatively, the slices may be scooped out continuously or discontinuously during sectioning and mounted symmetrically on glass slides at intervals of 1 tissue core diameter or 2 tissue core diameters.

[0014] Furthermore, it also includes:

[0015] The information content in the chip spotting organization is combined and arranged into a preset format according to the needs of the preset scene;

[0016] Style 1: More than 30 kinds of normal organ tissue microarray chips, including normal organ tissues of humans and animals;

[0017] Style 2: More than 10 normal organ tissue microarray chips are arranged according to a preset system, including normal organ tissues of humans and animals;

[0018] Style 3: Single or multiple normal organs arranged according to different functional parts of normal organ tissue microarray chips, including normal organ tissues of humans and animals;

[0019] The normal organ tissue is at least selected from the group consisting of esophagus, stomach, small intestine, large intestine, liver, pancreas, appendix, tongue, salivary gland, pharyngeal mucosa, lung, trachea, testicle, prostate, seminal vesicle, breast, ovary, endometrium, cervix, kidney, bladder, tonsil, lymph node, bone marrow, spleen, skin, artery, mesothelium, skeletal muscle, peripheral nerve, cerebellum, cerebrum, pituitary gland, adrenal gland, thyroid gland, parathyroid gland, and heart tissue.

[0020] Furthermore, the above-mentioned microarray chips of more than 30 kinds of normal organ tissues in Model 1 include:

[0021] Esophagus, stomach, small intestine, large intestine, liver, pancreas, appendix, tongue, salivary gland, pharyngeal mucosa, lung, testis, prostate, breast, ovary, endometrium, cervix, kidney, bladder, tonsil, lymph node, spleen, skin, artery, mesothelium, skeletal muscle, peripheral nerve, cerebellum, cerebrum, adrenal gland, thyroid gland, heart tissue, 3 cases for each organ.

[0022] Furthermore, the microarray chip of more than 10 kinds of normal organ tissues arranged in a systematic manner in the second style includes:

[0023] Brain, cerebellum, peripheral nerves, adrenal glands, thyroid gland, spleen, thymus, bone marrow, lymph nodes, tonsils, pancreas, liver, esophagus, stomach, small intestine, colon, lung, salivary gland, larynx, kidney, bladder, testis, prostate, vas deferens, ovary, fallopian tube, breast, endometrium, cervix, myocardium, eyeball, mesothelium, skin, 2 cases for each organ.

[0024] Furthermore, the single or multiple normal organs in the third style are arranged in accordance with different functional parts of the normal organ tissue microarray chip, including:

[0025] Mouse brain, cerebellum, spinal cord, hippocampus, eye, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, liver, gall bladder, pancreas, oral cavity, heart, artery, lung, trachea, kidney, ureter, bladder, prostate, testis, seminal vesicle, uterus, ovary, cervix, spleen, thymus, striated muscle, smooth muscle, skin, peripheral nerve, and fat. One to three marking points are added for each organ.

[0026] According to a second aspect of an embodiment of the present invention, a method for realizing a multi-element integrated multi-organ normal tissue microarray chip is provided, wherein the method for realizing a multi-element integrated multi-organ normal tissue microarray chip is applied to any of the multi-element integrated multi-organ normal tissue microarray chips described above, comprising:

[0027] Obtaining a tissue sample, and preprocessing the tissue sample to obtain a preprocessed tissue sample;

[0028] Dehydrating, transparentizing, wax-immersing and embedding the pretreated tissue sample to obtain a tissue wax block, and slicing the tissue wax block to obtain tissue wax block slices;

[0029] Performing HE staining and HE staining scanning on the tissue wax block slices to obtain a first processing result;

[0030] The tissue microarray chip is manufactured using the first processing result.

[0031] Furthermore, the obtaining of tissue samples and preprocessing of the tissue samples to obtain preprocessed tissue samples include:

[0032] The tissue sample is fixed in a 10% formalin fixative, and after 24 hours, the tissue is cut into tissues of corresponding sizes according to the tissue source to obtain a first tissue; wherein the volume of the fixative is 6 times greater than the volume of the specimen; human tissues are cut into a size of 1.5 cm×1.5 cm×0.5 cm, and rat and mouse organs are cut into a size of 1.0 cm×1.0 cm×0.5 cm; organs smaller than 1 cm are cut along the largest surface or completely flattened for fixation and embedding;

[0033] Fixing the first tissue with 10% formalin for 24 hours, and washing the fixed tissue block of the first tissue with running water for 24 hours to remove acid;

[0034] The deacidified tissue blocks were routinely dehydrated, transparentized, wax-impregnated, and sliced ​​to obtain pretreated tissue samples.

[0035] Furthermore, the pre-treated tissue sample is dehydrated, transparentized, wax-impregnated and embedded to obtain a tissue wax block, and the tissue wax block is sliced ​​to obtain tissue wax block slices, including:

[0036] The tissue wax block is sequentially immersed in alcohol of different concentrations at 37 degrees for corresponding time periods for dehydration treatment, then immersed in xylene I and II, and then immersed in different paraffin waxes at 60 degrees for different time periods for penetrating wax; wherein the alcohol concentrations include: 75%, 95%, 95%, 100%, 100% alcohol;

[0037] Using a preset embedding machine, the tissue wax block is clamped in according to corresponding requirements to determine the embedding direction, and after pressing on an embedding box of a specific specification, paraffin is injected, and the embedding is completed and frozen.

[0038] Furthermore, the step of using the first processing result to produce a tissue microarray chip comprises:

[0039] Mark effective target tissues and make tissue chip receptor wax blocks;

[0040] The effective target tissue area is marked on the control HE section, and a hole is punched on the tissue wax block to take samples;

[0041] The sample is taken out by a special instrument and embedded in the hole of the receptor wax block to obtain a tissue microarray wax block; wherein the special instrument includes: a tissue array instrument; the tissue microarray wax block is placed in a 54°C environment for 1 hour to solidify the wax and fuse, then placed at room temperature for 4 hours to adapt to the ambient temperature, and then transferred to a 4°C refrigerator for refrigeration for more than 6 hours for sectioning, baking and HE conventional staining to obtain a tissue microarray chip.

[0042] The technical solution provided by the embodiments of the present invention may have the following beneficial effects:

[0043] It is understandable that the technical solution provided by the present invention has the advantages of high throughput, high efficiency, parallelism, large amount of information and small error, and its application is becoming more and more extensive, not only suitable for histology and embryology, pathology teaching and use, but also suitable for immunohistochemistry, in situ hybridization, fluorescent immunoassay, PCR and other studies on the expression difference and comparison of target genes in different lesions (tumors) and normal tissues, the search for new genes for diseases (tumors) and drug screening, as well as a series of tests and controls such as disease pathology diagnosis and quality control. Achieve a more efficient, reliable, comprehensive and rigorous improvement of scientific research level and experimental model quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0045] Figure 1 is a schematic diagram of the structure of a multi-element integrated multi-organ normal tissue microarray chip according to an exemplary embodiment;

[0046] Figure 2The figure is a flow chart of a method for realizing a multi-element integrated multi-organ normal tissue microarray chip according to an exemplary embodiment. DETAILED DESCRIPTION

[0047] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are merely examples of devices and methods consistent with some aspects of the present invention as detailed in the appended claims.

[0048] Embodiment 1

[0049] See also Figure 1 , Figure 1 The figure is a schematic diagram of the structure of a multi-element integrated multi-organ normal tissue microarray chip according to an exemplary embodiment, wherein the chip comprises:

[0050] The multi-element integrated multi-organ normal tissue microarray chip is mainly composed of a glass slide substrate 1, a Zhongke Guanghua special label 3, and a tissue piece 2 with target information spots attached to the glass slide;

[0051] Wherein, the slide in the slide substrate 1 includes: a glass slide, a siliconized anti-shedding slide and a biofilm;

[0052] The target information dotted tissue slice 2 is carried on the glass slide substrate 1, and the distance between the tissue points of the target information dotted tissue corresponds to the preset tissue point diameter; wherein the diameter of the tissue points of the target information dotted tissue can be 0.6mm, 1.0mm, 1.5mm or 2.0mm; wherein the target information dotted tissue slice 2 carries a preset number of target information dotted tissues;

[0053] More specifically, the target information dotted tissue on the substrate can be 0.6mm in diameter (applicable to microarray tissue chips with high density of more than 300 dots, and the maximum arrangement of high density tissue dots on the standard glass slide is 702), 1.0mm (applicable to microarray tissue chips with medium-high density of 100-300 dots), 1.5mm (applicable to microarray tissue chips with medium-low density of 60-200 dots), and 2.0mm (applicable to microarray tissue chips with low density of less than 60 dots). The tissue dot spacing (row spacing or column spacing) is 0.2mm, 0.6mm (applicable to microarray tissue chips with high density of more than 300 dots), 0.3mm, 0.5mm (applicable to microarray tissue chips with medium-high density of 100-300 dots), 0.6mm, 0.8mm, 1.0mm, 1.5mm, 2.0mm, 3.0mm (applicable to microarray tissue chips with low density of less than 100 dots).

[0054] Among them, the target information dot organization on the slide base 1 is arranged from left to right in a preset row and column manner. From left to right in the horizontal direction, each row is numbered in sequence, and each column is numbered in alphabetical order in the vertical direction. There can be 702 points to 4 points on each base slide.

[0055] More specifically, from left to right, they are arranged in rows x columns, with each row numbered horizontally and each column numbered vertically, starting from 1-A. Each base slide can have 702 points to 4 points. The last point of the marking microarray is the last point, and the last point of the last row is the marking point (which can be a different tissue or a carbon core, etc.). This universal normal tissue (positive and / or negative) control microarray tissue chip includes various normal organs with effective target tissues, which can be applied to protein and molecular experiments (positive / negative) controls for various tumors and other diseases.

[0056] In specific implementation, the chips are arranged in rectangular matrix, square matrix or other shapes, and normal tissue control chips can be arranged in various combinations of microarrays according to preset requirements;

[0057] Among them, the row or column spacing can be 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.8mm, 1.0mm, 1.5mm, 2.0mm, 3.0mm;

[0058] Alternatively, the slices may be scooped out continuously or discontinuously during sectioning and mounted symmetrically on glass slides at intervals of 1 tissue core diameter or 2 tissue core diameters.

[0059] In the specific implementation, it also includes:

[0060] The information content in the chip spotting organization is combined and arranged into a preset format according to the needs of the preset scene;

[0061] Specifically, according to the needs of scientific research, teaching and clinical experiments, they can be combined and arranged into the following three styles: 1. More than 30 normal organ tissue microarray chips (including humans and animals); 2. More than 10 normal organ tissue microarray chips (including humans and animals) arranged according to a system; 3. Single (or multiple) normal organs and normal organ tissue microarray chips (including humans and animals) arranged according to different functional parts (≥1 functional part).

[0062] Style 1: More than 30 kinds of normal organ tissue microarray chips, including normal organ tissues of humans and animals;

[0063] Style 2: More than 10 normal organ tissue microarray chips are arranged according to a preset system, including normal organ tissues of humans and animals;

[0064] Style 3: Single or multiple normal organs arranged according to different functional parts of normal organ tissue microarray chips, including normal organ tissues of humans and animals;

[0065] The normal organ tissue is at least selected from the group consisting of esophagus, stomach, small intestine, large intestine, liver, pancreas, appendix, tongue, salivary gland, pharyngeal mucosa, lung, trachea, testicle, prostate, seminal vesicle, breast, ovary, endometrium, cervix, kidney, bladder, tonsil, lymph node, bone marrow, spleen, skin, artery, mesothelium, skeletal muscle, peripheral nerve, cerebellum, cerebrum, pituitary gland, adrenal gland, thyroid gland, parathyroid gland, and heart tissue.

[0066] In the specific implementation, please refer to Table 1 and Table 2. Table 1 is a microarray chip of more than 30 normal organ tissues in Style 1; Table 2 is a normal tissue chip of 33 organs, 3 cases for each organ.

[0067] Table 1

[0068]

[0069] Specifically, the more than 30 normal organ tissue microarray chips in style 1 include: esophagus, stomach, small intestine, large intestine, liver, pancreas, appendix, tongue, salivary gland, pharyngeal mucosa, lung, testicle, prostate, breast, ovary, endometrium, cervix, kidney, bladder, tonsil, lymph node, spleen, skin, artery, mesothelium, skeletal muscle, peripheral nerve, cerebellum, brain, adrenal gland, thyroid gland, heart tissue, 3 samples for each organ.

[0070] Table 2

[0071]

[0072]

[0073]

[0074]

[0075] In the specific implementation, please refer to Table 3 and Table 4. Table 3 is a microarray chip of more than 10 normal organ tissues arranged in a systematic manner in Style 2, and Table 4 is a combination chip of 33 normal organ tissues, with 2 examples for each organ.

[0076] Table 3

[0077]

[0078] During the specific implementation, it contains 33 normal human organs (cerebrum, cerebellum, peripheral nerves, adrenal glands, thyroid gland, spleen, thymus, bone marrow, lymph nodes, tonsils, pancreas, liver, esophagus, stomach, small intestine, colon, lungs, salivary glands, larynx, kidneys, bladder, testicles, prostate, vas deferens, ovaries, fallopian tubes, breasts, endometrium, cervix, myocardium, eyeballs, mesothelium, and skin), with 2 cases of each organ.

[0079] Table 4

[0080]

[0081]

[0082]

[0083]

[0084] During the specific implementation, please refer to Table 5 and Table 6. Table 5 is a normal organ tissue microarray chip in which a single or multiple normal organs in Style 3 are arranged according to different functional parts. Table 6 is a table of 36 normal organ tissue combination chips of mice.

[0085] Table 5

[0086]

[0087] Specifically, the normal organ tissue microarray chips of single or multiple normal organs in Style 3 are arranged according to different functional parts, including:

[0088] Mouse brain, cerebellum, spinal cord, hippocampus, eye, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, liver, gall bladder, pancreas, oral cavity, heart, artery, lung, trachea, kidney, ureter, bladder, prostate, testis, seminal vesicle, uterus, ovary, cervix, spleen, thymus, striated muscle, smooth muscle, skin, peripheral nerve, and fat. One to three marking points are added for each organ.

[0089] Table 6

[0090]

[0091]

[0092]

[0093]

[0094] In specific implementation, this series of tissue microarray chips has the advantages of high throughput, high efficiency, parallelism, large amount of information and small error. Its application is becoming more and more extensive. It is not only suitable for the teaching and use of tissue embryology and pathology, but also suitable for immunohistochemistry, in situ hybridization, fluorescent immunoassay, PCR and other studies on the expression differences and comparison of target genes in different lesions (tumors) and normal tissues, the search for new disease (tumor) genes and drug screening, as well as a series of tests and controls such as disease pathology diagnosis and quality control.

[0095] In specific implementation, the embodiment of the present application also provides a method for realizing a multi-element integrated multi-organ normal tissue microarray chip, and the method for realizing a multi-element integrated multi-organ normal tissue microarray chip is applied to any of the multi-element integrated multi-organ normal tissue microarray chips described above, comprising:

[0096] S1. Obtaining a tissue sample, pre-processing the tissue sample to obtain a pre-processed tissue sample;

[0097] S2. Dehydrating, transparentizing, wax-immersing and embedding the pretreated tissue sample to obtain a tissue wax block, and slicing the tissue wax block to obtain tissue wax block slices;

[0098] S3. Performing HE staining and HE staining scanning on the tissue wax block slices to obtain a first processing result;

[0099] S4. Using the first processing result, a tissue microarray chip is produced.

[0100] In a specific implementation, as shown in step S1, the obtaining of a tissue sample and preprocessing of the tissue sample to obtain a preprocessed tissue sample include:

[0101] The tissue sample is fixed in a 10% formalin fixative, and after 24 hours, the tissue is cut into tissues of corresponding sizes according to the tissue source to obtain a first tissue; wherein the volume of the fixative is 6 times greater than the volume of the specimen; human tissues are cut into a size of 1.5 cm×1.5 cm×0.5 cm, and rat and mouse organs are cut into a size of 1.0 cm×1.0 cm×0.5 cm; organs smaller than 1 cm are cut along the largest surface or completely flattened for fixation and embedding;

[0102] Fixing the first tissue with 10% formalin for 24 hours, and washing the fixed tissue block of the first tissue with running water for 24 hours to remove acid;

[0103] The deacidified tissue blocks were routinely dehydrated, transparentized, wax-impregnated, and sliced ​​to obtain pretreated tissue samples.

[0104] In a specific implementation, as described in step S2, the pre-treated tissue sample is dehydrated, transparent, wax-impregnated and embedded to obtain a tissue wax block, and the tissue wax block is sliced ​​to obtain tissue wax block slices, including:

[0105] The tissue wax block is sequentially immersed in alcohol of different concentrations at 37 degrees for corresponding time periods for dehydration treatment, then immersed in xylene I and II, and then immersed in different paraffin waxes at 60 degrees for different time periods for penetrating wax; wherein the alcohol concentrations include: 75%, 95%, 95%, 100%, 100% alcohol;

[0106] Using a preset embedding machine, the tissue wax block is clamped in according to corresponding requirements to determine the embedding direction, and after pressing on an embedding box of a specific specification, paraffin is injected, and the embedding is completed and frozen.

[0107] In the specific implementation, the process of dehydration, transparency, wax immersion and embedding into tissue wax blocks and observation under a section microscope (the melting point of Leica wax is 52°C-56°C) is as follows:

[0108] (1) 75% alcohol at 37 degrees for 2 hours;

[0109] (2) 95% alcohol at 37 degrees for 2 hours;

[0110] (3) 95% alcohol at 37 degrees for 2 hours;

[0111] (4) 100% alcohol at 37°C for 2 hours;

[0112] (5) 100% alcohol 37% for 1.5-2 hours;

[0113] (6) Xylene 1 45 minutes;

[0114] (7) Xylene II 45 minutes;

[0115] (8) Paraffin I at 60°C for 30 minutes;

[0116] (9) Paraffin II at 60°C for 60 minutes;

[0117] (10) Paraffin III at 60°C for 90 minutes;

[0118] Turn on the main power switch of the embedding machine two hours in advance, put the embedding machine in working state, increase the temperature, and let the embedding wax temperature reach about 58℃. Turn on the freezing table before embedding. Take out the embedding model, control the wax output by stepping on the foot, inject a small amount of paraffin, and clamp the tissue block. The tissue should be completely flattened. The embedding direction is determined according to the shape and anatomical structure of the tissue. For example, tubular and skin tissues should be embedded upright, and then the embedding box (5mm thickness specification) is pressed on. A small amount of paraffin is injected into the embedding box. After embedding, place it on the freezing table. When condensation appears on the surface of the embedding block on the freezing table, take out the wax block, trim off the surrounding excess wax, arrange it in order, and place it in the freezer of the refrigerator (-18℃) for use.

[0119] The HE (Hematoxylin-Eosin) staining steps are as follows: Fix the embedded wax block on the slicer and cut it into 3 micron thick wax slices. The thin wax slices cut continuously are often wrinkled and folded. Pull the thin wax slices at an angle of 25 to 30 degrees and put them in heated water (water temperature 45℃) to iron them flat, then stick them on the slide and put them in a 45℃ constant temperature box to dry for one hour.

[0120] (1) Insert the dried thin tissue slides into the staining rack one by one and gently place them;

[0121] (2) Xylene 1 for 5 minutes;

[0122] (3) Xylene II 10 minutes;

[0123] (4) anhydrous ethanol for 11 minutes;

[0124] (5) absolute ethanol II for 1 minute;

[0125] (6) 95% alcohol I1 minute;

[0126] (7) 95% alcohol II for 1 minute;

[0127] (8) 90% alcohol for 1 minute;

[0128] (9) 85% alcohol for 1 minute;

[0129] (10) Hematoxylin staining for 1 minute;

[0130] (11) Wash with tap water for 1 minute;

[0131] (12) 1% hydrochloric acid alcohol differentiation for 20 seconds;

[0132] (13) Wash with tap water for 1 minute;

[0133] (14) 1% ammonia water for blueing for 30 seconds;

[0134] (15) Wash with distilled water for 1 minute;

[0135] (16) Eosin staining for 1 minute;

[0136] (17) Wash with tap water for 1 minute;

[0137] (18) 85% alcohol for 1 minute;

[0138] (19) 90% alcohol for 1 minute;

[0139] (20) 95% alcohol for 1 minute;

[0140] (21) Anhydrous ethanol I 1 minute 30 seconds;

[0141] (22) Anhydrous ethanol II 2 minutes and 30 seconds;

[0142] (23) Xylene 1 for 1 minute;

[0143] (24) Xylene II 1 min;

[0144] (25) Neutral gum sealing;

[0145] (26) Staining results: The cell nucleus is blue and the cytoplasm is pink.

[0146] In a specific implementation, as described in step S3, HE staining and HE staining scanning are performed on the tissue wax block slices to obtain a first processing result, including the following steps:

[0147] HE (Hematoxylin-eosin staining) staining slide scan (40X magnification):

[0148] 1. A label is attached to the frosted area on the left side of the slide, which includes the logo, serial number, serial number and HE of Zhongke Guanghua;

[0149] 2. Place the scanning slide trays one by one and gently push them into the scanner (UNA PRECI CE 500B scanner);

[0150] 3. Automatically identify images and focus for scanning;

[0151] 4. Open the scanned image and observe that the scanned cells are clear and bright in color.

[0152] In a specific implementation, as described in step S4, the step of using the first processing result to produce a tissue microarray chip includes:

[0153] Mark effective target tissues and make tissue chip receptor wax blocks;

[0154] The effective target tissue area is marked on the control HE section, and a hole is punched on the tissue wax block to take samples;

[0155] The sample is taken out by a special instrument and embedded in the hole of the receptor wax block to obtain a tissue microarray wax block; wherein the special instrument includes: a tissue array instrument; the tissue microarray wax block is placed in a 54°C environment for 1 hour to solidify the wax and fuse, then placed at room temperature for 4 hours to adapt to the ambient temperature, and then transferred to a 4°C refrigerator for refrigeration for more than 6 hours for sectioning, baking and HE conventional staining to obtain a tissue microarray chip.

[0156] In the specific implementation, the microarray chip was prepared (using Beecher MTA-1 arrayer):

[0157] (1) The pathologist makes a diagnosis under a microscope based on the scan image and marks the effective target tissue;

[0158] (2) Make tissue chip receptor wax blocks (prepared according to the designed tissue microarray and spot size) and dig holes with a diameter of 0.3-2.0 mm on the blank wax block, with a hole spacing of 0.2 mm-2.0 mm, to make neatly arranged mold wax blocks;

[0159] (3) Punch holes in the donor wax block (tissue wax block) to sample the effective tissue area marked by the HE section;

[0160] (4) Take out the sample column core and bury it in the hole of the receptor wax block;

[0161] (5) After all the embedding is completed, place the tissue microarray wax block in a 54°C environment for 1 hour to solidify the wax and fuse it so that the tissue column core is fully compatible with the receptor wax block to prevent the spot from falling off;

[0162] (6) Place at room temperature for 4 hours to adapt to the ambient temperature, then transfer to a 4°C refrigerator and store for more than 6 hours for sectioning, baking, and HE conventional staining (the steps are the same as for large-section staining) to prepare a tissue microarray chip for scanning (the steps are the same as for section scanning);

[0163] (7) The pathologist observes the scanned image, magnifies it 50 to 400 times, and determines the diagnosis based on the tissue structure, cell morphology, and distribution within each tissue point; and evaluates the cell staining to determine the effective amount of tissue.

[0164] It can be understood that the same or similar parts of the above embodiments can be referenced to each other, and the contents not described in detail in some embodiments can refer to the same or similar contents in other embodiments.

[0165] It should be noted that, in the description of the present invention, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. In addition, in the description of the present invention, unless otherwise specified, the meaning of "plurality" refers to at least two.

[0166] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code that includes one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may not be performed in the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention belong.

[0167] It should be understood that the various parts of the present invention can be implemented by hardware, software, firmware or a combination thereof. In the above-mentioned embodiments, a plurality of steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented by hardware, as in another embodiment, it can be implemented by any one of the following technologies known in the art or their combination: a discrete logic circuit having a logic gate circuit for implementing a logic function for a data signal, a dedicated integrated circuit having a suitable combination of logic gate circuits, a programmable gate array (PGA), a field programmable gate array (FPGA), etc.

[0168] A person skilled in the art may understand that all or part of the steps in the method for implementing the above-mentioned embodiment may be completed by instructing related hardware through a program, and the program may be stored in a computer-readable storage medium, which, when executed, includes one or a combination of the steps of the method embodiment.

[0169] In addition, each functional unit in each embodiment of the present invention may be integrated into a processing module, or each unit may exist physically separately, or two or more units may be integrated into one module. The above-mentioned integrated module may be implemented in the form of hardware or in the form of a software functional module. If the integrated module is implemented in the form of a software functional module and sold or used as an independent product, it may also be stored in a computer-readable storage medium.

[0170] The storage medium mentioned above can be a read-only memory, a magnetic disk or an optical disk, etc.

[0171] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0172] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A multi-organ normal tissue microarray chip, characterized in that: The multi-element integrated multi-organ normal tissue microarray chip is mainly composed of a glass slide base, a Zhongke Guanghua special label, and a tissue piece with target information spots attached to the glass slide; Wherein, the slide in the slide substrate includes: a glass slide, a siliconized anti-shedding slide and a biofilm; The glass slide base carries a target information dotted tissue sheet, and the spacing between tissue points of the target information dotted tissue corresponds to a preset tissue point diameter; wherein the diameter of the tissue points of the target information dotted tissue can be 0.6 mm, 1.0 mm, 1.5 mm or 2.0 mm; wherein the target information dotted tissue sheet carries a preset number of target information dotted tissues; wherein the target information dotted tissues on the glass slide base are arranged from left to right in a preset row and column manner, from left to right horizontally, each row is numbered in sequence with numbers, and in the vertical direction, each column is numbered in alphabetical order, and each base glass slide can have 702 points to 4 points.

2. The multi-organ normal tissue microarray chip according to claim 1, characterized in that: The chips are arranged in rectangular matrix, square matrix or other shapes, and normal tissue control chips can be arranged in various combinations of microarrays according to preset requirements; Among them, the row or column spacing can be 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.8mm, 1.0mm, 1.5mm, 2.0mm, 3.0mm; Alternatively, the slices may be scooped out continuously or discontinuously during sectioning and mounted symmetrically on glass slides at intervals of 1 tissue core diameter or 2 tissue core diameters.

3. The multi-organ normal tissue microarray chip according to claim 1, characterized in that: Also includes: The information content in the chip spotting organization is combined and arranged into a preset format according to the needs of the preset scene; Style 1: More than 30 kinds of normal organ tissue microarray chips, including normal organ tissues of humans and animals; Style 2: More than 10 normal organ tissue microarray chips are arranged according to a preset system, including normal organ tissues of humans and animals; Style 3: Single or multiple normal organs arranged according to different functional parts of normal organ tissue microarray chips, including normal organ tissues of humans and animals; The normal organ tissue is at least selected from the group consisting of esophagus, stomach, small intestine, large intestine, liver, pancreas, appendix, tongue, salivary gland, pharyngeal mucosa, lung, trachea, testicle, prostate, seminal vesicle, breast, ovary, endometrium, cervix, kidney, bladder, tonsil, lymph node, bone marrow, spleen, skin, artery, mesothelium, skeletal muscle, peripheral nerve, cerebellum, cerebrum, pituitary gland, adrenal gland, thyroid gland, parathyroid gland, and heart tissue.

4. The multi-element integrated multi-organ normal tissue microarray chip according to claim 3, characterized in that: The above-mentioned 30 or more normal organ tissue microarray chips in Model 1 include: Esophagus, stomach, small intestine, large intestine, liver, pancreas, appendix, tongue, salivary gland, pharyngeal mucosa, lung, testis, prostate, breast, ovary, endometrium, cervix, kidney, bladder, tonsil, lymph node, spleen, skin, artery, mesothelium, skeletal muscle, peripheral nerve, cerebellum, cerebrum, adrenal gland, thyroid gland, heart tissue, 3 cases for each organ.

5. The multiplex integrated multi-organ normal tissue microarray chip according to claim 3, characterized in that: The microarray chips of more than 10 kinds of normal organ tissues arranged in a systematic manner in the second embodiment include: Brain, cerebellum, peripheral nerves, adrenal glands, thyroid gland, spleen, thymus, bone marrow, lymph nodes, tonsils, pancreas, liver, esophagus, stomach, small intestine, colon, lung, salivary gland, larynx, kidney, bladder, testis, prostate, vas deferens, ovary, fallopian tube, breast, endometrium, cervix, myocardium, eyeball, mesothelium, skin, 2 cases for each organ.

6. The multiplex integrated multi-organ normal tissue microarray chip according to claim 3, characterized in that: The single or multiple normal organs in the third style are arranged in accordance with different functional parts of the normal organ tissue microarray chip, including: Mouse brain, cerebellum, spinal cord, hippocampus, eye, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, liver, gall bladder, pancreas, oral cavity, heart, artery, lung, trachea, kidney, ureter, bladder, prostate, testis, seminal vesicle, uterus, ovary, cervix, spleen, thymus, striated muscle, smooth muscle, skin, peripheral nerve, and fat. One to three marking points are added for each organ.

7. A method for realizing a multi-element integrated multi-organ normal tissue microarray chip, characterized in that: The method for realizing the multi-element integrated multi-organ normal tissue microarray chip is applied to the multi-element integrated multi-organ normal tissue microarray chip according to any one of claims 1 to 6, comprising: Obtaining a tissue sample, and preprocessing the tissue sample to obtain a preprocessed tissue sample; Dehydrating, transparentizing, wax-immersing and embedding the pretreated tissue sample to obtain a tissue wax block, and slicing the tissue wax block to obtain tissue wax block slices; Performing HE staining and HE staining scanning on the tissue wax block slices to obtain a first processing result; The tissue microarray chip is manufactured using the first processing result.

8. The method for realizing the multi-element integrated multi-organ normal tissue microarray chip according to claim 7, characterized in that: The step of obtaining a tissue sample and preprocessing the tissue sample to obtain a preprocessed tissue sample includes: The tissue sample is fixed in a 10% formalin fixative, and after 24 hours, the tissue is cut into tissues of corresponding sizes according to the tissue source to obtain a first tissue; wherein the volume of the fixative is 6 times greater than the volume of the specimen; human tissues are cut into a size of 1.5 cm×1.5 cm×0.5 cm, and rat and mouse organs are cut into a size of 1.0 cm×1.0 cm×0.5 cm; organs smaller than 1 cm are cut along the largest surface or completely flattened for fixation and embedding; Fixing the first tissue with 10% formalin for 24 hours, and washing the fixed tissue block of the first tissue with running water for 24 hours to remove acid; The deacidified tissue blocks were routinely dehydrated, transparentized, wax-impregnated, and sliced ​​to obtain pretreated tissue samples.

9. The method for realizing the multi-element integrated multi-organ normal tissue microarray chip according to claim 7, characterized in that: The method of dehydrating, transparentizing, wax-immersing and embedding the pretreated tissue sample to obtain a tissue wax block, and slicing the tissue wax block to obtain tissue wax block slices comprises: The tissue wax block is sequentially immersed in alcohol of different concentrations at 37 degrees for corresponding time periods for dehydration treatment, then immersed in xylene I and II, and then immersed in different paraffin waxes at 60 degrees for different time periods for penetrating wax; wherein the alcohol concentrations include: 75%, 95%, 95%, 100%, 100% alcohol; Using a preset embedding machine, the tissue wax block is clamped in according to corresponding requirements to determine the embedding direction, and after pressing on an embedding box of a specific specification, paraffin is injected, and the embedding is completed and frozen.

10. The method for realizing the multi-element integrated multi-organ normal tissue microarray chip according to claim 7, characterized in that: The method of using the first processing result to produce a tissue microarray chip comprises: Mark effective target tissues and make tissue chip receptor wax blocks; The effective target tissue area is marked on the control HE section, and a hole is punched on the tissue wax block to take samples; The sample is taken out by using a special instrument and embedded in the hole of the receptor wax block to obtain a tissue microarray wax block; wherein the special instrument includes: a tissue array instrument; The tissue microarray wax block was placed in a 54°C environment for 1 hour to solidify the wax and fuse, then placed at room temperature for 4 hours to adapt to the ambient temperature, and then transferred to a 4°C refrigerator for more than 6 hours for sectioning, baking and HE conventional staining to obtain a tissue microarray chip.

Images