Immunohistochemical enzyme-labeled immunosorbent method and kit for use in the immunohistochemical enzyme-labeled immunosorbent method
By simplifying the immunohistochemistry steps and solution preparation, combined with specific markers and blockers, the existing immunohistochemistry methods are solved, and the problems of complex operation and unstable staining effect are achieved, making it easy to operate, controllable staining effect and reduced cost. effect.
Patent Information
- Application Number
- CN202011619526.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-12-30
- Publication Date
- 2025-01-14
- Estimated Expiration
- 2040-12-30
AI Technical Summary
The existing immunohistochemistry methods are complex in operation, unstable staining effect, and unreasonable dosage and equipment, which leads to inconvenient use and high cost.
A series of simplified steps and solution preparation methods are adopted, including paraffin section baking, dewaxing, antigen repair, blocking, labeled antibody dropping and color development reaction, using 0.3% hydrogen peroxide in methanol solution as a blocker, CD276 antibody , biotin-labeled goat anti-mouse IgG and streptomyces avidin-peroxidase and other markers, the desired solvent is provided through a kit packaged in the drop bottle.
The operation is simplified, the accuracy and stability of the dyeing effect are ensured, the problems of insufficient or excessive doses are avoided, the production cost is reduced, and the convenience of use is improved.
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, in particular to an immunohistochemical enzyme-labeled immunosorbent method and a kit used for the immunohistochemical enzyme-labeled immunosorbent method. Background Art
[0002] Immunohistochemistry is the application of the basic principle of immunology - antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody. It uses chemical reaction to make the color developer (fluorescein, enzyme, metal ion, isotope) labeled with antibody develop color to determine the antigen (peptide and protein) in tissue cells, and conduct localization, qualitative and relative quantitative research on it. This is called immunohistochemistry or immunocytochemistry.
[0003] Immunohistochemistry has important functional significance for tumor pathological diagnosis and basic morphological research. This method is used to detect normal or abnormal products within or between cells, locate protein components, explore pathogenesis, determine the occurrence and type of certain tumor tissues, and screen markers with clinical potential.
[0004] Immunohistochemistry technology includes two aspects: first, the recognition system, that is, specific antibodies recognize antigens in tissues or cells; second, the display system, that is, the specific binding of antigens and antibodies is displayed through a microscope and becomes a morphological reaction visible to the naked eye. Immunohistochemistry technology can be divided into immunofluorescence, immunoenzyme, immunoferritin, immunogold and radioimmunoautography according to the type of marker. Among them, immunoenzyme is the most commonly used technology in immunohistochemistry research. The basic principle is to first react enzyme-labeled antibodies with tissues or cells, and then add enzyme substrates to generate colored insoluble products or particles with a certain electron density. The corresponding antigens in cells or tissues are located or qualitatively studied by light microscopy or electron microscopy.
[0005] However, the current immunohistochemistry methods are complicated to operate, the staining effect is extremely unstable, the preparation methods of each dye or solution are also extremely complicated, the production cost is high, and the dosage is unreasonable. During use, insufficient or excessive dosage may occur, causing inconvenience in use. Summary of the invention
[0006] In order to overcome the defects in the prior art, the present invention provides an immunohistochemical enzyme labeling method, comprising the following steps:
[0007] The paraffin sections were sliced and placed in an oven at 60°C for 1 h;
[0008] The baked slices were placed in xylene for dewaxing, and the process was repeated 3 times, each time for 5 minutes;
[0009] The dewaxed sections were sequentially placed in anhydrous ethanol for 1 min, 95% ethanol for 30 s, and 75% ethanol for 30 s, then rinsed in tap water for 1-2 min, and then rinsed with PBS 3 times, 3 min each time;
[0010] The dehydrated slices were immersed in 0.01M citrate buffer, microwave repaired for 5 minutes, cooled naturally to room temperature, and then replaced with citrate buffer and microwave repaired for 5 minutes;
[0011] After antigen retrieval, the sections were placed in a blocking agent, incubated at room temperature for 20 min, and rinsed with PBS three times, each time for 3 min;
[0012] The slices were spread in a wet box, 100ul of blocking solution was added, incubated at room temperature for 15-20min, washed with PBS 3 times, 3min each time, and a waterproof circle was drawn on the slice 3-5mm outside the tissue with an immunohistochemistry pen to prevent the liquid from drying up;
[0013] The sections were spread in a wet box, 50-100 ul of primary antibody was added, incubated overnight in a refrigerator at 4°C, and rinsed with PBS 3 times, 3 min each time;
[0014] Add 50-100 ul of biotin-labeled secondary antibody to the slices, incubate at room temperature for 15-20 min, and rinse with PBS three times, each time for 3 min;
[0015] Add 50-100ul of avidin-labeled HRP to the slices, incubate at room temperature for 15-20min, and rinse with PBS three times, each time for 3min;
[0016] 100ul DAB color developing solution was added to the slices and color was developed for 5-10min. During the period, the tissue was observed under a microscope. If the tissue showed positive results, the reaction could be terminated in tap water.
[0017] Hematoxylin was added to the sections for counterstaining for 30 seconds, and the sections were washed with water to return to blue.
[0018] After the blueing is completed, the slices are placed in 75% ethanol for 1 min, 95% ethanol for 1 min, anhydrous ethanol for 1 min, and xylene twice, each for 1 min;
[0019] The alcohol-dehydrated slices were sealed with neutral gum and dried naturally.
[0020] Furthermore, the blocking agent is a 0.3% methanol solution of hydrogen peroxide.
[0021] Furthermore, the primary antibody is CD276 antibody, and the CD276 antibody is diluted, and the antibody dilution ratio is 1:200.
[0022] Furthermore, the biotin-labeled secondary antibody is biotin-labeled goat anti-mouse IgG.
[0023] Furthermore, the avidin-labeled HRP is streptavidin-peroxidase.
[0024] Furthermore, the blocker is an endogenous peroxidase blocker, and the preparation method of the endogenous peroxidase blocker comprises: slowly adding 150 μl of 30% hydrogen peroxide into 15 ml of anhydrous methanol solution and mixing thoroughly.
[0025] Furthermore, the method for preparing the blocking solution includes: adding 600 mg of bovine serum albumin to 14.8 ml of phosphate buffer, and adding 200 μl of 30% Triton after fully dissolving.
[0026] Furthermore, the method for preparing the biotin-labeled goat anti-mouse IgG includes: adding 150 mg of bovine serum albumin to 14.8 ml of phosphate buffer, and then adding 200 μl of 30% Triton and 1 μl of storage solution.
[0027] Furthermore, the preparation method of the streptavidin-peroxidase comprises: preparing a storage solution by cross-linking 0.2 mg of streptavidin with 0.8 mg of horseradish peroxidase and dissolving them in 1 ml of deionized water; and adding 75 μl of the storage solution into 15 ml of phosphate buffer.
[0028] Furthermore, the present invention also includes a kit for the immunoenzyme labeling method, comprising:
[0029] Endogenous peroxidase blocker, including: 150 μl of 30% hydrogen peroxide and 15 ml of anhydrous methanol solution;
[0030] High-efficiency blocking solution, including: 600 mg of bovine serum albumin, 14.8 ml of phosphate buffer, and 200 μl of 30% Triton;
[0031] Biotinylated goat anti-mouse IgG, including: 150 mg bovine serum albumin, 14.8 ml phosphate buffer, 200 μl 30% Triton and 1 μl storage solution;
[0032] Streptavidin-peroxidase includes: 75 μl of storage solution and 15 ml of phosphate buffer, wherein the storage solution is formed by cross-linking 0.2 mg of streptavidin and 0.8 mg of horseradish peroxidase and dissolving in 1 ml of deionized water.
[0033] The beneficial effects of the present invention are as follows:
[0034] The immunohistochemical immunoenzyme labeling method of the present invention is simple to operate, and the color can be accurately controlled by microscopic examination to avoid over-staining and ensure the staining effect. The preparation method of each solution is simple, the dosage is suitable, and the use is convenient to avoid waste.
[0035] The present invention also provides a kit, which includes the solvents required by the above-mentioned immunoenzyme labeling method, and each solvent is packaged in a dropper bottle, which is convenient to use.
[0036] In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail as follows. DETAILED DESCRIPTION
[0037] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0038] This embodiment relates to an immunohistochemical enzyme-labeled immunosorbent method, comprising the following steps:
[0039] Paraffin blank sections were sliced and placed in an oven at 60°C for 1 h;
[0040] The baked slices were placed in xylene for dewaxing, and the process was repeated 3 times, each time for 5 minutes;
[0041] The dewaxed sections were sequentially placed in anhydrous ethanol for 1 min, 95% ethanol for 30 s, and 75% ethanol for 30 s, then rinsed in tap water for 1-2 min, and then rinsed with PBS 3 times, 3 min each time;
[0042] The dehydrated slices were immersed in 0.01M citrate buffer, microwave repaired for 5 minutes, cooled naturally to room temperature, and then replaced with citrate buffer and microwave repaired for 5 minutes;
[0043] After antigen retrieval, the sections were placed in a blocking agent, incubated at room temperature for 20 min, and rinsed with PBS three times, each time for 3 min;
[0044] The slices were spread in a wet box, 100ul of blocking solution was added, incubated at room temperature for 15-20min, washed with PBS 3 times, 3min each time, and a waterproof circle was drawn on the slice 3-5mm outside the tissue with an immunohistochemistry pen to prevent the liquid from drying up;
[0045] The sections were spread in a wet box, 50-100 ul of primary antibody was added, incubated overnight in a refrigerator at 4°C, and rinsed with PBS 3 times, 3 min each time;
[0046] Add 50-100 ul of biotin-labeled secondary antibody to the slices, incubate at room temperature for 15-20 min, and rinse with PBS three times, each time for 3 min;
[0047] Add 50-100ul of avidin-labeled HRP to the slices, incubate at room temperature for 15-20min, and rinse with PBS three times, each time for 3min;
[0048] 100ul DAB color developing solution was added to the slices and color was developed for 5-10min. During the period, the tissue was observed under a microscope. If the tissue showed positive results, the reaction could be terminated in tap water.
[0049] Hematoxylin was added to the sections for counterstaining for 30 seconds, and the sections were washed with water to return to blue.
[0050] After the blueing is completed, the slices are placed in 75% ethanol for 1 min, 95% ethanol for 1 min, anhydrous ethanol for 1 min, and xylene twice, each for 1 min;
[0051] The alcohol-dehydrated slices were sealed with neutral gum and dried naturally.
[0052] The protein of the paraffin sections stained by the immunoenzyme labeling method of this embodiment is brown or brownish yellow.
[0053] Wherein, the PBS in the immunoenzyme labeling method is 0.01M phosphate buffer (PH7.4).
[0054] The blocking agent in the immunoenzyme labeling method is a methanol solution of 0.3% hydrogen peroxide. The blocking agent is used as an endogenous peroxidase blocking agent, and its preparation method includes: slowly adding 150 μl of 30% hydrogen peroxide to 15 ml of anhydrous methanol solution and fully mixing.
[0055] The blocking solution in the immunoenzyme labeling method is a high-efficiency blocking solution, and its preparation method comprises: adding 600 mg of bovine serum albumin to 14.8 ml of phosphate buffer, and adding 200 μl of 30% Triton after fully dissolving.
[0056] The primary antibody in the immunoenzyme labeling method is CD276 antibody, and the CD276 antibody is diluted, and the antibody dilution ratio is 1:200.
[0057] The biotin-labeled secondary antibody in the immunoenzyme labeling method is biotin-labeled goat anti-mouse IgG. The preparation method of biotin-labeled goat anti-mouse IgG includes: adding 150 mg bovine serum albumin to 14.8 ml phosphate buffer, then adding 200 μl of 30% Triton and 1 μl of storage solution.
[0058] The avidin-labeled HRP in the immunoenzyme labeling method is streptavidin-peroxidase. The preparation method of streptavidin-peroxidase includes: cross-linking 0.2 mg of streptavidin with 0.8 mg of horseradish peroxidase, dissolving in 1 ml of deionized water to form a storage solution; adding 75 μl of the storage solution to 15 ml of phosphate buffer.
[0059] The immunohistochemical immunoenzyme labeling method is easy to operate, and the color can be accurately controlled through microscopic examination to avoid over-staining and ensure the staining effect. The preparation method of each solution is simple, the dosage is suitable, and the use is convenient to avoid waste.
[0060] The present invention also includes a kit for the immunoenzyme labeling method, comprising:
[0061] Endogenous peroxidase blocker, including: 150 μl of 30% hydrogen peroxide and 15 ml of anhydrous methanol solution;
[0062] High-efficiency blocking solution, including: 600 mg of bovine serum albumin, 14.8 ml of phosphate buffer, and 200 μl of 30% Triton;
[0063] Biotinylated goat anti-mouse IgG, including: 150 mg bovine serum albumin, 14.8 ml phosphate buffer, 200 μl 30% Triton and 1 μl storage solution;
[0064] Streptavidin-peroxidase includes: 75 μl of storage solution and 15 ml of phosphate buffer, wherein the storage solution is formed by cross-linking 0.2 mg of streptavidin and 0.8 mg of horseradish peroxidase and dissolving in 1 ml of deionized water.
[0065] Among them, the endogenous peroxidase blocker, high-efficiency blocking solution, biotin-labeled goat anti-mouse IgG and streptavidin-peroxidase in the kit are respectively packaged in a dropper bottle, each of which contains 15 ml of liquid.
[0066] The kit includes solvents required for the immunoenzyme labeling method, and each solvent is packaged in dropper bottles for easy use.
[0067] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.
Claims
1. An immunohistochemical enzyme-labeled method, characterized in that: The steps include: The paraffin sections were sliced and placed in an oven at 60°C for 1 h; The baked slices were placed in xylene for dewaxing, and the process was repeated 3 times, each time for 5 minutes; The dewaxed sections were sequentially placed in anhydrous ethanol for 1 min, 95% ethanol for 30 s, and 75% ethanol for 30 s, then rinsed in tap water for 1-2 min, and then rinsed with PBS 3 times, 3 min each time; The dehydrated slices were immersed in 0.01M citrate buffer, microwave repaired for 5 minutes, cooled naturally to room temperature, and then replaced with citrate buffer and microwave repaired for 5 minutes; The sections after antigen repair are placed in a blocking agent, incubated at room temperature for 20 minutes, and rinsed with PBS three times, each time for 3 minutes; wherein the blocking agent is a 0.3% hydrogen peroxide methanol solution; the blocking agent is an endogenous peroxidase blocker, and the preparation method of the endogenous peroxidase blocker comprises slowly adding 150 μl of 30% hydrogen peroxide to 15 ml of anhydrous methanol solution and mixing thoroughly; The slices were flattened in a wet box, 100ul of blocking solution was added, incubated at room temperature for 15-20min, rinsed with PBS 3 times, 3min each time, and a waterproof circle was drawn on the slice 3-5mm outside the tissue with an immunohistochemistry pen to prevent the liquid from draining; wherein the method for preparing the blocking solution comprises adding 600mg of bovine serum albumin to 14.8ml of phosphate buffer, and adding 200μl of 30% Triton after fully dissolving; The slices were flattened in a wet box, 50-100ul of primary antibody was added, incubated in a refrigerator at 4 degrees overnight, and washed with PBS 3 times, each time for 3 minutes; wherein the primary antibody was CD276 antibody, and the CD276 antibody was diluted, and the antibody dilution ratio was 1:200; Add 50-100ul of biotin-labeled secondary antibody to the slice, incubate at room temperature for 15-20min, and rinse with PBS 3 times, each time for 3min, wherein the biotin-labeled secondary antibody is biotin-labeled goat anti-mouse IgG, which is prepared by adding 150mg of bovine serum albumin to 14.8ml of phosphate buffer, then adding 200μl of 30% Triton and 1μl of storage solution; Add 50-100ul of avidin-labeled HRP to the slice, incubate at room temperature for 15-20min, and rinse with PBS 3 times, each time for 3min, wherein the avidin-labeled HRP is streptavidin-peroxidase, and its preparation method is to cross-link 0.2mg of streptavidin with 0.8mg of horseradish peroxidase, and dissolve in 1ml of deionized water to form a storage solution; add 75μl of the storage solution into 15ml of phosphate buffer; 100ul DAB color developing solution was added to the slices and color was developed for 5-10min. During the period, the tissue was observed under a microscope. If the tissue showed positive results, the reaction could be terminated in tap water. Add hematoxylin to the sections for counterstaining for 30 seconds, and wash with water to return to blue; After the blueing is completed, the slices are placed in 75% ethanol for 1 min, 95% ethanol for 1 min, anhydrous ethanol for 1 min, and xylene twice, each for 1 min; The alcohol-dehydrated slices were sealed with neutral gum and dried naturally.