Dewaxing, repairing and blocking integrated body fluid and application thereof in pathological specimen immunohistochemical operation method

Abstract

The invention provides dewaxing, repairing and blocking integrated liquid and application thereof in a pathological specimen immunohistochemical operation method. The dewaxing, repairing and blocking integrated liquid is prepared from the following components in percentage by mass: 1.0 to 5.0 percent of Triton-100, 1.0 to 5.0 percent of Tween 20, 0.1 to 3.0 percent of fatty acid methyl ester ethoxylate, 0.1 to 1.0 percent of tetrahydrofurfuryl alcohol, 0.001 to 0.01 percent of 2-methyl-3-isothiazolinone, 0.01 to 4.0 percent of buffer solution and the balance of water. In the process of applying the dewaxing, repairing and blocking integrated body fluid to the pathological specimen immunohistochemical operation method, compared with an existing laboratory method, the dewaxing, repairing and blocking integrated body fluid is used at the temperature of 65-75 DEG C, the steps of xylene dewaxing and gradient alcohol hydration after dewaxing in an existing laboratory can be replaced, the temperature continues to rise to 95-99 DEG C, and when antigen repairing is conducted on a sample, the temperature of the sample is lowered to 95-99 DEG C; according to the method, the step of blocking 0.3-3% of hydrogen peroxide in an existing laboratory is replaced by the enzyme activity of high temperature on endogenous catalase, and the same effects of dewaxing, repairing and blocking in the existing laboratory are achieved on the basis of reducing the experimental steps and the experimental time.

Description

Technical Field

[0001] The present invention relates to the technical field of laboratory test analysis, and particularly to a dewaxing, repair and blocking integrated solution and its application in the immunohistochemical operation method of pathological specimens. Background Technique

[0002] Immunohistochemical experiments play an important role in biomedical diagnosis and related research. A complete immunohistochemical experiment process includes steps such as dewaxing, antigen repair, endogenous bioenzyme blocking, antibody incubation, chromogenic reaction of enzyme-labeled chromogenic agent, and counterstaining. Different steps need to be washed with tris(hydroxymethyl)aminomethane buffer (TBS buffer) or phosphate buffer (PBS buffer) before the next operation can be carried out. Therefore, there are many steps, which greatly reduces the work efficiency.

[0003] During the immunohistochemical experiment process, antigen repair and endogenous catalase blocking are considered to be one of the key steps affecting the immunohistochemical staining effect. Antigen repair treatment is used to expose antigenic determinants, promote the specific binding reaction of antigen and antibody, and improve the visibility of its signal. The traditional method believes that the blocking of endogenous catalase is used to reduce background signals and reduce false positives. For endogenous catalase, it widely exists in a variety of cells and can catalyze the decomposition of hydrogen peroxide to produce water and oxygen. When detecting the immunohistochemical chromogenic reaction system labeled with horseradish peroxidase, if no proper treatment is carried out, endogenous catalase will react with the chromogenic agent, resulting in non-specific background staining or false positives, thus interfering with and affecting the experimental results. The basic method for antigen repair is heat-induced repair method, and the blocking of endogenous catalase is carried out with 0.3% - 3% hydrogen peroxide. In enzyme kinetics, temperature and pH are two important factors affecting enzyme activity. Most enzymes denature and inactivate above 60°C. Therefore, the heat repair treatment at 99°C can denature or inactivate the endogenous catalase protein, thereby reducing non-specific background staining. However, in the currently commonly used immunohistochemical experiments, even when repairing under high heat conditions, hydrogen peroxide is still used for blocking endogenous catalase. Whether it is necessary to add hydrogen peroxide reagent for blocking endogenous catalase after treatment by the heat repair method has not been proposed in relevant literatures or technical guidelines at present. Summary of the Invention

[0004] (I) Technical Problems to be Solved

[0005] Aiming at the deficiencies of the prior art, the present invention provides a dewaxing, repair and blocking integrated solution and its application in the immunohistochemical operation method of pathological specimens. By using a specific dewaxing, repair and blocking integrated solution, the conditions of the dewaxing, repair and blocking steps are adjusted to realize the one-step operation of dewaxing, repair and blocking, and solve the problems put forward in the above background technique.

[0006] (2) Technical Solution

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] According to the first aspect of the present invention, a dewaxing, repair and blocking integrated liquid is provided. By mass fraction, it includes 1.0 - 5.0% of Triton-100, 1.0 - 5.0% of Tween 20, 0.1 - 3.0% of fatty acid methyl ester ethoxylate, 0.1 - 1.0% of tetrahydrofurfuryl alcohol, 0.001 - 0.01% of 2-methyl-3-isothiazolinone, 0.01 - 4.0% of buffer solution and the balance of water.

[0009] Preferably, the buffer solution includes TE buffer solution or citric acid - sodium citrate buffer solution;

[0010] The TE buffer solution includes 0.01 - 1.0% of EDTA and 0.01 - 1.0% of Tris-HCl;

[0011] The citric acid - sodium citrate buffer solution includes 0.1 - 1% of citric acid and 1.0 - 3.0% of sodium citrate.

[0012] According to the second aspect of the present invention, a method for immunohistochemistry operation of pathological specimens is provided. Using the above dewaxing, repair and blocking integrated liquid, it includes the following steps:

[0013] 1) Sample pretreatment: Section the paraffin sample and bake it at 75°C for 1 - 5 h;

[0014] 2) Dewaxing, repair and blocking: Immerse the section in the dewaxing, repair and blocking integrated liquid preheated to 65 - 75°C for 5 - 10 min, and then continue to heat up to 95 - 99°C and hold for 20 min; or drop the dewaxing, repair and blocking integrated liquid on the section, heat it at 75°C for dewaxing for 5 - 10 min, elute with TBST buffer solution, re-drop the dewaxing, repair and blocking integrated liquid, heat it at 95 - 99°C for repair for 20 min, and finally elute with TBST buffer solution;

[0015] 3) Primary antibody incubation: Incubate at room temperature for 30 - 60 min, and after incubation, elute with TBST buffer solution;

[0016] 4) Secondary antibody incubation: Incubate at room temperature for 25 - 30 min, and after incubation, elute with TBST buffer solution;

[0017] 5) DAB color development: Add DAB color developer, incubate at room temperature for 5 min, and after color development, elute with pure water;

[0018] 6) Hematoxylin counterstaining: Incubate with hematoxylin at room temperature for 2 - 60 s, and after color development, elute with pure water;

[0019] 7) Sealing the section.

[0020] Preferably, in step 1), the thickness of the section is 3 - 5 μm.

[0021] Preferably, in step 3), the loading volume of the primary antibody incubation is 100 - 150 μL.

[0022] Preferably, in step 4), the loading volume of the secondary antibody incubation is 100 - 150 μL.

[0023] Preferably, in step 5), the loading volume of the DAB chromogenic agent is 100 - 150 μL.

[0024] Preferably, in step 6), the loading volume of the hematoxylin counterstaining is 100 - 150 μL.

[0025] (III) Beneficial effects

[0026] The present invention provides a dewaxing, repair and blocking integrated solution and its application in the immunohistochemistry operation method of pathological specimens. It has the following beneficial effects:

[0027] The dewaxing, repair and blocking integrated solution provided by this scheme and its application in the immunohistochemistry operation method of pathological specimens, compared with the existing laboratory methods, uses a specific dewaxing, repair and blocking integrated solution at 65 - 75 °C, which can replace the steps of dewaxing with xylene and gradient alcohol hydration after dewaxing in the existing laboratory. Then continue to heat up to 95 - 99 °C. While performing antigen repair on the sample, the enzyme activity of endogenous catalase is inactivated by high temperature, replacing the 0.3 - 3% hydrogen peroxide blocking step in the existing laboratory. It realizes the same effect as dewaxing, repair and blocking in the existing laboratory on the basis of reducing experimental steps and experimental time. Description of the drawings

[0028] Figure 1 It is the detection result of TTF-1 immunohistochemistry on thyroid tissue in Example 1 of the present invention;

[0029] Figure 2 It is the detection result of TTF-1 immunohistochemistry on thyroid tissue in Comparative Example 1 of the present invention;

[0030] Figure 3 It is the detection result of CD3 immunohistochemistry on tonsil tissue in Example 2 of the present invention;

[0031] Figure 4 It is the detection result of CD3 immunohistochemistry on tonsil tissue in Comparative Example 2 of the present invention;

[0032] Figure 5Detection results of P504S immunohistochemistry on renal tissue in Example 3 of the present invention;

[0033] Figure 6 Detection results of P504S immunohistochemistry on renal tissue in Comparative Example 3 of the present invention;

[0034] Figure 7 Verification diagram of whether there is endogenous catalase in the yeast of the present invention;

[0035] Figure 8 Verification diagram of the blocking of endogenous catalase by the hydrogen peroxide blocking time of the present invention;

[0036] Figure 9 Comparison diagram of the effect of high temperature on the activity of endogenous catalase of the present invention;

[0037] Figure 10 Comparison diagram of the effect of sample pre-fixation on the activity of endogenous catalase of the present invention;

[0038] Figure 11 IHC comparison diagram of human tonsil tissue under different repair and blocking conditions of the present invention;

[0039] Figure 12 Color development comparison diagram of serositis gastric tissue containing red blood cells of the present invention. Detailed implementation manners

[0040] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. The suitable implementation manners and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0041] Verification description of a single step in the process of immunohistochemistry operation method for pathological specimens of the present invention.

[0042] First, the requirements and pre-treatment preparation process for the proof experiment samples are as follows:

[0043] Yeast-like slices: In a 0.5 mL EP tube, add water to the dry yeast powder at a ratio of 1:1 and place it at room temperature for 30 min for yeast rehydration; drop the rehydrated yeast onto the glass slide and dry the slide at 30 °C for 10 min, and moisten it with PBS buffer before the experiment;

[0044] Tonsil samples: Select wax blocks of chronic suppurative tonsil tissue, section them at a thickness of 4 μm, pick up the sections, dry them, and then bake them in an oven at 75 °C.

[0045] Serositis gastric tissue-like slices: Select wax blocks of serositis gastric tissue with bleeding, section them at a thickness of 4 μm, pick up the sections, dry them, and then bake them in an oven at 75 °C.

[0046] I. Verification of the existence of endogenous catalase in yeast:

[0047] (1) Under the conditions of no heating and no hydrogen peroxide blockage, a complete DAB color development experiment was carried out using DAB chromogenic substrate and DAB buffer; the color development results are as shown in Figure A of Figure 7 . It can be seen that the number of colored yeast samples is rich, and the bacterial cells are colored brownish-yellow, indicating that a complete DAB color development experiment has been completed.

[0048] (2) Under the conditions of no heating and no hydrogen peroxide blockage, only the DAB chromogenic substrate was added, and DAB buffer (hydrogen peroxide) was not used for the DAB color development experiment; the color development results are as shown in Figure B of Figure 7 . It can be seen that the yeast cells show blue coloration with hematoxylin and no brownish-yellow coloration, indicating that the DAB color reaction is incomplete.

[0049] It can be proved by Figure A and Figure B of Figure 7 that in the DAB reaction process, when hydrogen peroxide is lacking in the DAB reaction system, the catalytic effect of catalase in the DAB reaction cannot be reflected.

[0050] II. Comparison of the effect of hydrogen peroxide blocking time on the blocking of endogenous catalase:

[0051] (1) Under the conditions of no heating and blocked with 3% hydrogen peroxide for 5 minutes, a DAB color development experiment was carried out on yeast, and the color development results are as shown in Figure A of Figure 8 . The yeast cells of the yeast sample show blue coloration with hematoxylin and no brownish-yellow coloration.

[0052] (2) Under the conditions of no heating and blocked with 3% hydrogen peroxide for 10 minutes, a DAB color development experiment was carried out on yeast, and the color development results are as shown in Figure B of Figure 8 . The yeast cells of the yeast sample show blue coloration with hematoxylin and no brownish-yellow coloration.

[0053] It can be seen that the endogenous catalase of yeast without high-temperature treatment cannot catalyze the DAB color reaction after being blocked with 3% hydrogen peroxide for different times.

[0054] III. Comparison of the effect of high temperature on the activity of endogenous catalase:

[0055] (1) Under the conditions of no heating and no hydrogen peroxide blockage, a DAB color development experiment was carried out on yeast; as shown in Figure A of Figure 9 , the number of colored yeast samples is rich, and the bacterial cells are colored brownish-yellow.

[0056] (2) Yeast cells were subjected to DAB color development experiments under heating at 80 °C without hydrogen peroxide blocking; as shown in Figure B of Figure 9 , only the yeast cell bodies in the yeast sample showed blue staining with hematoxylin, without brownish-yellow staining.

[0057] It can be seen that after high-temperature treatment of yeast, its endogenous catalase has been inactivated and cannot catalyze the DAB color development reaction.

[0058] IV. Comparison of the effects of sample pre-fixation on the activity of endogenous catalase:

[0059] (1) Yeast cells were subjected to DAB color development experiments under fixation with 95% ethanol, without heating, and without hydrogen peroxide blocking; the color development results are as shown in Figure A of Figure 10 , and some of the yeast cell bodies in the yeast sample showed brownish-yellow staining.

[0060] (2) Yeast cells were subjected to DAB color development experiments under fixation with 4% neutral formaldehyde, without heating, and without hydrogen peroxide blocking; the color development results are as shown in Figure B of Figure 10 , and some of the yeast cell bodies in the yeast sample showed brownish-yellow staining.

[0061] (3) Yeast cells were subjected to DAB color development experiments without fixation, without heating, and without hydrogen peroxide blocking; the color development results are as shown in Figure C of Figure 10 , and the number of stained yeast cells was abundant, and the cell bodies showed brownish-yellow staining.

[0062] It can be seen that after fixation, the activity of endogenous catalase in yeast has been partially inactivated, and its catalytic effect on the DAB color development reaction has weakened.

[0063] V. IHC comparison of human tonsil tissues under different repair and blocking conditions:

[0064] (1) Tonsil tissues were subjected to immunohistochemical experiments of the immune marker CK under antigen heat repair and blocking with 3% hydrogen peroxide; the color development results are as shown in Figure A of Figure 11 , CK was normally expressed, and no non-specific staining background was produced.

[0065] (2) Tonsil tissues were subjected to immunohistochemical experiments of the immune marker CK under antigen heat repair without hydrogen peroxide blocking; the color development results are as shown in Figure B of Figure 11 , CK was normally expressed, and no non-specific staining background was produced.

[0066] (3) Tonsil tissues were subjected to immunohistochemical experiments of the immune marker CK without antigen heat repair and blocking with 3% hydrogen peroxide; the color development results are as shown in Figure C of Figure 11 , CK could not be expressed, and no brownish-yellow background was produced.

[0067] (4) The tonsil tissue was subjected to immunohistochemical experiment of the immune marker CK under the conditions of without antigen heat repair and without hydrogen peroxide blocking; the color development result is as shown in Figure D of Figure 11 , CK could not be expressed, and a weak brownish-yellow background was produced.

[0068] It can be seen that in the tonsil tissue after antigen repair, its endogenous catalase has been inactivated, which can effectively reduce the generation of non-specific staining background.

[0069] VI. Color development experiment of serositis gastric tissue containing red blood cells:

[0070] (1) After dewaxing the gastric tissue, hematoxylin-eosin (H-E) staining was performed; the color development experiment result is as shown in Figure A of 12, the cell nuclei were stained blue, the cytoplasm was stained light red, and the red blood cells were stained red. This example sample had serositis and bleeding;

[0071] (2) After dewaxing and blocking the gastric tissue, hematoxylin counterstaining was directly performed (without antibody incubation and DAB color development); the color development experiment result is as shown in Figure 12 Figure B of, only the cell nuclei were stained blue, and the red blood cells had no color development;

[0072] (3) After dewaxing the gastric tissue (without blocking), hematoxylin counterstaining was directly performed (without antibody incubation and DAB color development); the color development experiment result is as shown in Figure 12 Figure C of, the cell nuclei were stained blue, and the red blood cells were stained light yellow;

[0073] (4) After dewaxing and blocking the gastric tissue, DAB color development was performed first (without antibody incubation), and then hematoxylin counterstaining was performed; the color development experiment result is as shown in Figure 12 Figure D of, the cell nuclei were stained blue, and the red blood cells had no color development;

[0074] (5) After dewaxing the gastric tissue (without blocking), DAB color development was performed first (without antibody incubation), and then hematoxylin counterstaining was performed; the color development experiment result is as shown in Figure 12 Figure E of, the cell nuclei were stained blue, and the red blood cells were stained brownish-yellow;

[0075] (6) After dewaxing, repairing and blocking the gastric tissue, immunohistochemical detection of the Desmin immune marker was performed; the detection result is as shown in Figure 12 Figure F of, in the nucleated cell area, the cell nuclei were stained blue, the cytoplasm was stained brownish-yellow, and the red blood cells had no color development;

[0076] (7) After dewaxing and repairing the gastric tissue (without blocking), immunohistochemical detection of the Desmin immune marker was performed; the detection result is as shown in Figure 12As shown in Figure G, in the area of nucleated cells, the cell nuclei are stained blue, the cytoplasm is stained brownish-yellow, and the red blood cells are stained light yellow;

[0077] (8) After dewaxing the gastric tissue (without repair and without blocking), immunohistochemical detection of Desmin immunoreagent was performed; the detection results are as Figure 12 shown in Figure H, in the area of nucleated cells, the cell nuclei are stained blue, the cytoplasm is stained brownish-yellow, and the red blood cells are stained brownish-yellow;

[0078] Through the color development experiments on the serositis gastric tissue containing red blood cells: color development experiments (1), (2), (3), it is suggested that the blocking agent (hydrogen peroxide) has a bleaching effect on red blood cells; color development experiments (4), (5), it is suggested that red blood cells not bleached by the blocking agent (hydrogen peroxide) will be stained brownish-yellow by DAB, that is, red blood cells contain endogenous catalase; color development experiments (6), (7), (8), it is suggested that in immunohistochemical experiments, if after repair, whether blocked or not, red blood cells cannot react with DAB for color development, that is, the endogenous catalase in red blood cells has been inactivated by heat repair.

[0079] The following uses human tissues to compare the technical solutions of the present invention according to the implementation steps.

[0080] Example 1

[0081] An operation method for performing TTF-1 immunohistochemistry on thyroid tissue, comprising the following steps:

[0082] 1) Slicing: Slicing the paraffin sample of thyroid tissue into slices of 4 μm, and baking at 75 °C for 2 h;

[0083] 2) Integrated dewaxing, repair, and blocking treatment: Immerse the slices in the integrated dewaxing, repair, and blocking solution preheated to 75 °C, then continue to heat to 99 °C and maintain for 20 min, and finally elute with TBST buffer solution, wherein the integrated dewaxing, repair, and blocking solution includes 1.0% Triton-100, 1.0% Tween 20, 0.1% fatty acid methyl ester ethoxylate, 0.1% tetrahydrofurfuryl alcohol, 0.001% 2-methyl-3-isothiazolinone, 0.01% EDTA, 0.01% Tris-HCl, and the balance of water;

[0084] 3) Primary antibody incubation: Incubate at room temperature for 45 min, the sample loading amount is 100 μL, and after incubation, elute with TBST buffer solution;

[0085] 4) Secondary antibody incubation: Incubate at room temperature for 30 min, the sample loading amount is 100 μL, and after incubation, elute with TBST buffer solution;

[0086] 5) DAB Chromogenic Reaction: Add DAB chromogenic reagent and incubate at room temperature for 5 min. The sample loading volume is 100 μL. After the chromogenic reaction is completed, wash it with pure water.

[0087] 6) Hematoxylin Counterstaining: Incubate with hematoxylin at room temperature for 30 s. After the chromogenic reaction is completed, wash it with pure water.

[0088] 7) Sealing the Slide: Seal the slide with a mounting medium.

[0089] The detection results of TTF-1 immunohistochemistry on thyroid tissue in Example 1 are as Figure 1 shown.

[0090] Comparative Example 1

[0091] 1) Sample Pretreatment: Cut the paraffin sample of thyroid tissue into sections with a thickness of 4 μm and bake at 75 °C for 2 h.

[0092] 2) Dewaxing and Hydration Treatment: Immerse the sections in xylene I and xylene II for 5 min respectively for dewaxing, and then immerse them in absolute ethanol I, absolute ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol once each for 5 min. After completion, wash them with TBST buffer.

[0093] 3) Antigen Retrieval: Adopt the heat retrieval method to heat and retrieve the sections in EDTA antigen retrieval buffer at a temperature of 99 °C for 20 min. The sample loading volume is 100 mL. After the retrieval is completed, wash them with TBST buffer.

[0094] 4) Catalase Blocking: Drop 1% catalase blocker to block endogenous catalase and incubate at room temperature for 5 min. The sample loading volume is 100 μL. After blocking, wash it with TBST buffer.

[0095] 5) Primary Antibody Incubation: Incubate at room temperature for 45 min. The sample loading volume is 100 μL. After incubation, wash it with TBST buffer.

[0096] 6) Secondary Antibody Incubation: Incubate at room temperature for 30 min. The sample loading volume is 100 μL. After incubation, wash it with TBST buffer.

[0097] 7) DAB Chromogenic Reaction: Add DAB chromogenic reagent and incubate at room temperature for 5 min. The sample loading volume is 100 μL. After the chromogenic reaction is completed, wash it with pure water.

[0098] 8) Hematoxylin Counterstaining: Incubate with hematoxylin at room temperature for 30 s. After the chromogenic reaction is completed, wash it with pure water.

[0099] 9) Sealing the Slide: Seal the slide with a mounting medium.

[0100] The detection results of TTF-1 immunohistochemistry on thyroid tissue using conventional laboratory methods in this comparative example are as follows Figure 2 shown. According to the comparison between Figure 1 and Figure 2 , it can be proved that the dewaxing, repair, and blocking one-step method of the present invention can achieve the same effect as the existing conventional laboratory dewaxing, repair, and blocking methods.

[0101] Example 2

[0102] An operation method for CD3 immunohistochemistry on tonsil tissue includes the following steps:

[0103] 1) Sectioning: Section the paraffin sample of tonsil tissue into sections of 4 μm and bake at 75 °C for 2 h;

[0104] 2) Integrated dewaxing, repair, and blocking treatment: Drop the dewaxing, repair, and blocking integrated solution onto the sections, heat for dewaxing at 75 °C for 10 min, after elution, continue to heat to 99 °C and hold for 20 min, and finally elute with TBST buffer solution. Among them, the dewaxing, repair, and blocking integrated solution includes 1.0% Triton-100, 1.0% Tween 20, 0.1% fatty acid methyl ester ethoxylate, 0.1% tetrahydrofurfuryl alcohol, 0.001% 2-methyl-3-isothiazolinone, 0.01% EDTA, 0.01% Tris-HCl, and the balance of water;

[0105] 3) Primary antibody incubation: Incubate at room temperature for 45 min, the sample loading amount is 100 μL, and after incubation, elute with TBST buffer solution;

[0106] 4) Secondary antibody incubation: Incubate at room temperature for 30 min, the sample loading amount is 100 μL, and after incubation, elute with TBST buffer solution;

[0107] 5) DAB color development: Add DAB color developer, incubate at room temperature for 5 min, the sample loading amount is 100 μL, and after color development, elute with pure water;

[0108] 6) Hematoxylin counterstaining: Incubate with hematoxylin at room temperature for 30 s, and after color development, elute with pure water;

[0109] 7) Sealing: Seal with a sealing agent.

[0110] The detection results of CD3 immunohistochemistry on tonsil tissue in this example are as follows Figure 3 shown.

[0111] Comparative Example 2

[0112] 1) Sample pretreatment: Section the paraffin sample of tonsil tissue into sections of 4 μm and bake at 75 °C for 2 h;

[0113] 2) Dewaxing and hydration treatment: The sections were immersed in xylene I and xylene II for 5 minutes respectively for dewaxing, and then successively immersed in absolute ethanol I, absolute ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each time. After completion, they were eluted with TBST buffer;

[0114] 3) Antigen repair: Using the heat repair method, add EDTA antigen repair buffer to the sections, at a temperature of 99 °C, repair for 20 minutes, the sample addition volume is 150 mL, and after repair, it is eluted with TBST buffer;

[0115] 4) Catalase blocking: Add 1% catalase blocker to block endogenous catalase, incubate at room temperature for 5 minutes, the sample addition volume is 100 μL, and after blocking, it is eluted with TBST buffer;

[0116] 5) Primary antibody incubation: Incubate at room temperature for 45 minutes, the sample addition volume is 100 μL, and after incubation, it is eluted with TBST buffer;

[0117] 6) Secondary antibody incubation: Incubate at room temperature for 30 minutes, the sample addition volume is 100 μL, and after incubation, it is eluted with TBST buffer;

[0118] 7) DAB color development: Incubate at room temperature for 30 minutes, the sample addition volume is 100 μL, and after incubation, it is eluted with TBST buffer;

[0119] 8) Hematoxylin counterstaining: Incubate with hematoxylin at room temperature for 30 seconds, and after color development, it is eluted with pure water;

[0120] 9) Sealing: Seal with a sealing agent.

[0121] The detection results of CD3 immunohistochemistry on tonsil tissue in this comparative example using the conventional laboratory method are as Figure 4 shown. According to the comparison between Figure 3 and Figure 4 it can be proved that the dewaxing, repair, and blocking one-step method of the present invention can achieve the same effect as the existing conventional laboratory dewaxing, repair, and blocking methods.

[0122] Example 3

[0123] An operation method for P504S immunohistochemistry on kidney tissue, including the following steps:

[0124] 1) Sectioning: Section the paraffin sample of kidney tissue into sections of 4 μm and bake at 75 °C for 2 hours;

[0125] 2) Integrated dewaxing, repair, and blocking treatment: Drop the integrated dewaxing, repair, and blocking solution onto the sections, heat for dewaxing at 75°C for 10 min. After elution, continue to heat to 99°C and maintain for 20 min. Finally, elute with TBST buffer. Among them, the integrated dewaxing, repair, and blocking solution includes 1.0% Triton-100, 1.0% Tween 20, 0.1% fatty acid methyl ester ethoxylate, 0.1% tetrahydrofurfuryl alcohol, 0.001% 2-methyl-3-isothiazolinone, 1% citric acid, 2.0% sodium citrate, and the balance of water;

[0126] 3) Primary antibody incubation: Incubate at room temperature for 45 min, the sample loading volume is 100 μL. After incubation, elute with TBST buffer;

[0127] 4) Secondary antibody incubation: Incubate at room temperature for 30 min, the sample loading volume is 100 μL. After incubation, elute with TBST buffer;

[0128] 5) DAB color development: Add DAB color developer, incubate at room temperature for 5 min, the sample loading volume is 100 μL. After color development, elute with pure water;

[0129] 6) Hematoxylin counterstaining: Incubate with hematoxylin at room temperature for 30 s. After color development, elute with pure water;

[0130] 7) Sealing: Seal with a sealing agent.

[0131] The detection results of P504S immunohistochemistry on renal tissue in this example are as Figure 5 shown.

[0132] Comparative Example 2

[0133] 1) Sample pretreatment: Cut the paraffin sample of renal tissue into sections with a thickness of 4 μm, and bake at 75°C for 2 h;

[0134] 2) Dewaxing and hydration treatment: Immerse the sections in xylene I and xylene II for 5 min respectively for dewaxing, and then immerse them in absolute ethanol I, absolute ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol once each for 5 min. After completion, elute with TBST buffer;

[0135] 3) Antigen repair: Adopt the heat repair method, drop the EDTA antigen repair buffer onto the sections, the temperature is 99°C, repair for 20 min, the sample loading volume is 150 mL. After repair, elute with TBST buffer;

[0136] 4) Catalase blocking: Drop 1% catalase blocker to block endogenous catalase, incubate at room temperature for 5 min, the sample loading volume is 100 μL. After blocking, elute with TBST buffer;

[0137] 5) Primary antibody incubation: Incubate at room temperature for 45 min with a sample volume of 100 μL. After incubation, wash with TBST buffer.

[0138] 6) Secondary antibody incubation: Incubate at room temperature for 30 min with a sample volume of 100 μL. After incubation, wash with TBST buffer.

[0139] 7) DAB color development: Incubate at room temperature for 30 min with a sample volume of 100 μL. After incubation, wash with TBST buffer.

[0140] 8) Hematoxylin counterstaining: Incubate with hematoxylin at room temperature for 30 s. After color development, wash with pure water.

[0141] 9) Sealing: Seal with a mounting medium.

[0142] The detection results of P504S immunohistochemistry on renal tissue using the conventional method in the laboratory in this comparative example are as Figure 6 shown. According to the comparison between Figure 5 and Figure 6 , it can be proved that the dewaxing, repair, and blocking one-step method of the present invention can achieve the same effect as the existing conventional dewaxing, repair, and blocking methods in the laboratory.

[0143] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A dewaxing, repairing and blocking integrated liquid, characterized in that: The invention comprises, by mass fraction, 1.0-5.0% of Triton-100, 1.0-5.0% of Tween 20, 0.1-3.0% of fatty acid methyl ester ethoxylate, 0.1-1.0% of tetrahydrofurfuryl alcohol, 0.001-0.01% of 2-methyl-3-isothiazolinone, 0.01-4.0% of buffer solution and the balance of water.

2. A dewaxing, repairing and blocking integrated liquid according to claim 1, characterized in that: The buffer comprises TE buffer or citric acid-sodium citrate buffer; The TE buffer comprises 0.01-1.0% EDTA and 0.01-1.0% Tris-HCl; The citric acid-sodium citrate buffer solution comprises 0.1-1% citric acid and 1.0-3.0% sodium citrate.

3. A method for immunohistochemistry of pathological specimens, characterized in that: The dewaxing, repairing and blocking integrated liquid according to claim 1 or claim 2 is used, comprising the following steps: 1) Sample pretreatment: slice the paraffin samples and bake at 75°C for 1 to 5 hours; 2) Dewaxing, repairing, and blocking: immersing the slices in a dewaxing, repairing, and blocking integrated solution preheated to 65-75° C. for 5-10 minutes, and then continuing to heat to 95-99° C. and maintaining for 20 minutes; or dripping a dewaxing, repairing, and blocking integrated solution on the slices, heating and dewaxing at 75° C. for 5-10 minutes, eluting with TBST buffer, dripping a dewaxing, repairing, and blocking integrated solution again, heating and repairing at 95-99° C. for 20 minutes, and finally eluting with TBST buffer; 3) Primary antibody incubation: incubate at room temperature for 30-60 min, and then elute with TBST buffer; 4) Secondary antibody incubation: incubate at room temperature for 25-30 minutes, and then elute with TBST buffer; 5) DAB color development: Add DAB color developing agent, incubate at room temperature for 5 minutes, and wash with pure water after color development; 6) Hematoxylin staining: Incubate with hematoxylin at room temperature for 2 to 60 seconds, and wash with pure water after color development; 7) Seal the slides.

4. The method for immunohistochemistry of pathological specimens according to claim 3, characterized in that: In step 1), the thickness of the slice is 3 to 5 μm.

5. The method for immunohistochemistry of pathological specimens according to claim 3, characterized in that: In step 3), the volume of the primary antibody incubation is 100-150 μL.

6. The method for immunohistochemistry of pathological specimens according to claim 3, characterized in that: In step 4), the volume of the secondary antibody incubation is 100-150 μL.

7. The method for immunohistochemistry of pathological specimens according to claim 3, characterized in that: In step 5), the amount of the DAB developer added is 100-150 μL.

8. The method for immunohistochemistry of pathological specimens according to claim 3, characterized in that: In step 6), the amount of hematoxylin staining added is 100-150 μL.

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