Dewaxing repair liquid for immunohistochemical detection

Abstract

The dewaxing repair liquid is prepared from the following components in parts by mass: 10 to 20 parts of a water-soluble dewaxing agent, 0.1 to 0.5 part of an antigen repair agent, 10 to 20 parts of a tissue clearing agent, 0.1 to 0.5 part of a tissue penetrating agent, 0.5 to 1 part of a PH (Potential of Hydrogen) stabilizer and the balance of water. The dewaxing repair liquid disclosed by the invention can be used for directly carrying out antigen repair without dewaxing and hydration in advance, so that the operation steps are shortened, the experiment time is saved, and the effect is not obviously different from that of step-by-step dewaxing, hydration and repair.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay detection, and in particular relates to a dewaxing repair solution for immunohistochemical detection. Background Art

[0002] In short, immunohistochemistry is a common pathological test method that uses known antibodies to detect antigens in the tissue to be tested. Immunohistochemistry can not only help determine the type, nature and origin of the tumor, but also be used for treatment guidance and prognosis of lesions, which can make many difficult tumors clearly diagnosed. Tissues are usually fixed to preserve antigens and prevent autolysis or necrosis of the obtained tissues. Embedded tissues can provide continuous support during the sectioning process, making the sections more durable. The most commonly used and main test sample type for immunohistochemistry is pathological paraffin tissue sections. However, since paraffin-embedded tissue sections are water-insoluble and the antigenic determinants in the tissues are blocked, it is difficult for antibodies to fully bind to antigens and cannot be directly used for immunohistochemical detection. Before immunohistochemical detection, the sample needs to be pretreated. The repair solution can restore the function of the antigenic determinant or expose the antigenic determinant site, so that the antibody can bind to it and prepare for the next step of staining and labeling, that is, dewaxing, hydration, and antigen repair are required.

[0003] Traditional tissue wax block sections need to be dewaxed, hydrated, and repaired, which are complex, time-consuming, and labor-intensive. In addition, xylene is required, which is toxic and harmful to operators and the environment. Therefore, it is of great significance to improve the dewaxing and repairing solution and its operation steps. Summary of the invention

[0004] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a dewaxing repair solution for immunohistochemistry detection, which can directly perform antigen repair without the need for dewaxing and hydration in advance, shortening the operation steps, saving experimental time, and the effect is no significantly different from the dewaxing, hydration and repair step by step.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: A dewaxing repair solution for immunohistochemical detection comprises the following components in parts by mass: 10 to 20 parts of a water-soluble dewaxing agent, 0.1 to 0.5 parts of an antigen repair agent, 10 to 20 parts of a tissue transparent agent, 0.1 to 0.5 parts of a tissue penetrant, 0.5 to 1 parts of a pH stabilizer, and the remainder being water.

[0006] Preferably, the water-soluble dewaxing agent is one or more of polyethylene glycol-200, polypropylene glycol-400, polyethylene glycol-20000 and Triton X-100, and the volume fraction of the water-soluble dewaxing agent is 20%.

[0007] Preferably, the antigen retrieval agent is EDTA at a concentration of 1 mM.

[0008] Preferably, the tissue clearing agent is 20% propylene glycol by volume.

[0009] Preferably, the tissue penetrant is sodium dodecyl sulfate with a mass fraction of 0.01%.

[0010] Preferably, the aforementioned pH stabilizer is a Tris-HCl buffer with a pH of 9.0 and a concentration of 25 mM.

[0011] The preparation method of the dewaxing repair liquid comprises the following steps: adding a tissue penetrant, a tissue transparent agent and an antigen repair agent to a pH stabilizer, stirring evenly, then adding a water-soluble dewaxing agent, stirring and dissolving sufficiently, and mixing evenly to prepare the dewaxing repair liquid.

[0012] The antigen repair method using the above dewaxing repair solution comprises the following steps: S1. Dewaxing and repairing: adding tissue paraffin sections into dewaxing and repairing solution, dewaxing and repairing, and drying; S2, blocking: add hydrogen peroxide blocking agent to the dried slices and incubate in the dark; S3, primary antibody incubation: add primary antibody reagent to the tissue site and incubate at a constant temperature; S4, secondary antibody incubation: add secondary antibody reagent to the tissue site and incubate at room temperature; S5, color development: add DAB working solution to the slices and incubate at room temperature in the dark; S6, counterstaining-dehydration-sealing: add hematoxylin staining solution to the slices, incubate at room temperature, dehydrate with gradient ethanol solution, dry in the air, and obtain neutral gum sealing slides; S7. Slide review: Observe the staining of the coverslips.

[0013] The benefits of the present invention are as follows: the dewaxing and repairing solution of the present invention does not use organic solvents such as xylene and alcohol, and is more environmentally friendly; the present invention integrates the operating steps of dewaxing, hydration, and antigen repair of tissue sections, and directly performs antigen repair without the need for dewaxing and hydration in advance, thereby shortening the operating steps, saving experimental time, and the effect is not significantly different from that of dewaxing, hydrating, and repairing in separate steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the IHC staining effect diagram of different water-soluble dewaxing agents with the same content in Example 1 ( Figure 1 A: Polyethylene glycol-200, Figure 1 B: Polypropylene glycol-400, Figure 1 C: Polyethylene glycol-20000, Figure 1 D: Triton TX-100, Figure 1E: polyethylene glycol-200); Figure 2 This is the IHC staining effect diagram of different contents of tissue clearing agent in Example 2 ( Figure 2 A. Figure 2 B. Figure 2 C is 5%, 10%, and 20% by volume of glycerol, respectively); Figure 3 This is a comparison diagram of the IHC staining effect of dewaxing, hydration, and repair in Example 3 ( Figure 3 A: The effect diagram of IHC staining with the dewaxing repair solution of the present invention in one step, Figure 3 B: IHC staining effect diagram of traditional dewaxing, hydration and repair step by step). DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] Example 1: Comparison of different water-soluble dewaxing agents with the same content A dewaxing repair solution for immunohistochemical detection comprises the following components: 10-20 parts of a water-soluble dewaxing agent, 0.1-0.5 parts of an antigen repair agent (EDTA), 10-20 parts of a tissue transparent agent (propylene glycol), 0.1-0.5 parts of a tissue penetrant (sodium dodecyl sulfate), 0.5-1 parts of a pH stabilizer (Tris-Hcl (pH 9.0)), and the remainder is water; the water-soluble dewaxing agent is selected from one of polyethylene glycol-200, polypropylene glycol-400, polyethylene glycol-20000 and Triton X-100, and the specific components are shown in Table 1.

[0017] Table 1 Components of dewaxing repair fluid

[0018] The dewaxing repair solution preparation method comprises the following steps: 1. Weigh 3.03 g of Tris, fully dissolve it in 1 L of pure water, and use hydrochloric acid to adjust the pH to 9.0 to prepare a Tris-HCl buffer solution; 2. Add sodium dodecyl sulfate, propylene glycol, and EDTA to the Tris-HCl buffer solution in sequence according to the composition ratio in Table 1, and use four water-soluble dewaxing agents to fully stir and dissolve, mix well, and prepare four dewaxing repair solutions with different water-soluble dewaxing agents.

[0019] The prepared dewaxing repair solution was used for antigen repair and the staining effect was observed. The results are shown in Figure 1 The antigen repair method includes the following steps: (1) Dewaxing and repairing: Place the tissue paraffin sections in a 65°C oven for 40 minutes, add dewaxing and repairing solution and dewax and repair at 102°C for 11 minutes, then repeat the process of adding dewaxing and repairing solution and dewax and repair at 102°C for 11 minutes, rinse the tissue sections from top to bottom with TBS buffer, and repeat the rinse three times; (2) Blocking: Add 80-100 μL of hydrogen peroxide blocking agent to the dried slices, incubate at room temperature in the dark for 10 min, and then rinse the tissue slices from top to bottom with TBS buffer for 3 times; (3) Primary antibody incubation: Add 1-2 drops of primary antibody reagent (about 50-100 μL) to the tissue site; observe that the tissue is completely immersed in the primary antibody reagent, and then incubate at 37°C for 1 hour; when the time is up, take it out of the incubator and observe the incubation status; rinse the tissue section from top to bottom with TBS buffer, and repeat the rinse 3 times; (4) Secondary antibody incubation: wipe off the residual TBS buffer and add 1-2 drops of secondary antibody reagent (about 50-100 μL) to the tissue site; observe that the tissue is completely immersed in the secondary antibody reagent and incubate at room temperature for 25 minutes (room temperature 22-26°C is appropriate); when the time is up, rinse the tissue section from top to bottom with TBS buffer and repeat 3 times; (5) Color development: Add 80-100 μL of freshly prepared DAB working solution to the slices and incubate at room temperature in the dark for 5-10 min. (6) Restaining - dehydration - sealing: Wash off the excess DAB working solution with purified water, then add 10-30 μL of hematoxylin staining solution, incubate at room temperature for 10-30 seconds, then use 75%, 95%, and 100% ethanol solutions for gradient dehydration for 3 minutes each, air dry, and finally obtain neutral gum sealing slides; (7) Film review: Observe the staining conditions under a microscope.

[0020] Depend on Figure 1 It can be seen that Figure 1 A and 1B analysis show that the water-soluble dewaxing agent polyethylene glycol-200 has a stronger dyeing effect than polypropylene glycol-400, and polyethylene glycol-200 is preferred; Figure 1 The results of analysis of C, 1D, and 1E show that the water-soluble dewaxing agents polyethylene glycol-20000 and Triton TX-100 have weaker staining effects than polyethylene glycol-200, and a higher degree of background staining. Polyethylene glycol-200 (where Figure 1 A and 1B are the same tissue and target. Figure 1 C, 1D, and 1E are the same tissues and targets).

[0021] Example 2: Comparison of different amounts of tissue clearing agents A dewaxing and repairing solution for immunohistochemical detection includes the following components: a water-soluble dewaxing agent (polyethylene glycol-200), an antigen repair agent (EDTA), a tissue transparent agent (glycerol), a tissue penetrant (sodium dodecyl sulfate), and a pH stabilizer (Tris-Hcl (pH 9.0)), wherein the volume fraction of glycerol added is different. The specific components are shown in Table 2.

[0022] Table 2 Components of dewaxing repair fluid

[0023] The prepared dewaxing repair solution was used for antigen repair and the staining effect was observed. The results are shown in Figure 2 .Depend on Figure 2 A, 2B, and 2C analyses showed that for the same tissue clearing agent glycerol at different contents, the group with a volume fraction of 20% had a higher staining intensity and a lower background coloring than the groups with a volume fraction of 5% and 10%. Therefore, the tissue clearing agent is preferably glycerol with a volume fraction of 20%.

[0024] Example 3: Comparison between the antigen dewaxing and repairing methods of the present invention and the conventional dewaxing and repairing methods Dewaxing and repairing solution were prepared according to the components in Table 3, and the antigen repair method in Example 1 was used for staining. The staining effect was compared with the traditional method of dewaxing, hydration and repairing step by step. The results are shown in Figure 3 .

[0025] Table 3 Components of dewaxing repair fluid

[0026] The dewaxing, hydration and fixing step-by-step method for immunohistochemistry is as follows: Compared with the method for using the dewaxing repair solution in Example 1, only step (1) is different, which is changed to: place the tissue paraffin slices in a 65°C oven for 40 minutes, soak the tissue slices in 3 cylinders of xylene in sequence, soaking each cylinder for 3 minutes; soak the tissue slices in anhydrous ethanol → 95% ethanol → 75% ethanol in sequence, soaking each cylinder for 3 minutes; add the dewaxing repair solution to dewax and repair at 102°C for 11 minutes, and then repeat the dewaxing repair solution at 102°C for 11 minutes. Rinse the tissue slices from top to bottom with TBS buffer, and repeat the rinsing 3 times.

[0027] Depend on Figure 3 A and 3B analysis showed that the dewaxing and repairing solution of the present invention was comparable to the traditional dewaxing, hydration and repairing step-by-step method in terms of IHC staining intensity and background coloring degree.

[0028] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A dewaxing repair solution for immunohistochemical detection, characterized in that: The invention comprises the following components in parts by weight: 10 to 20 parts of a water-soluble dewaxing agent, 0.1 to 0.5 parts of an antigen repair agent, 10 to 20 parts of a tissue transparent agent, 0.1 to 0.5 parts of a tissue penetrant, 0.5 to 1 parts of a pH stabilizer, and the rest is water.

2. The dewaxing repair solution for immunohistochemical detection according to claim 1, characterized in that: The water-soluble dewaxing agent is one or more of polyethylene glycol-200, polypropylene glycol-400, polyethylene glycol-20000 and Triton X-100, and the volume fraction of the water-soluble dewaxing agent is 20%.

3. The dewaxing repair solution for immunohistochemical detection according to claim 1, characterized in that: The antigen retrieval agent is EDTA with a concentration of 1 mM.

4. The dewaxing repair solution for immunohistochemical detection according to claim 1, characterized in that: The tissue clearing agent is 20% propylene glycol by volume.

5. The dewaxing repair solution for immunohistochemical detection according to claim 1, characterized in that: The tissue penetrant is sodium dodecyl sulfate with a mass fraction of 0.01%.

6. The dewaxing repair solution for immunohistochemical detection according to claim 1, characterized in that: The pH stabilizer is a Tris-HCl buffer solution with a pH of 9.0 and a concentration of 25 mM.

7. The method for preparing the dewaxing repair liquid according to claim 1, characterized in that: The following steps are involved: Add tissue penetrant, tissue transparent agent and antigen repair agent to the pH stabilizer, stir evenly, then add water-soluble dewaxing agent, stir thoroughly to dissolve, and mix well to prepare dewaxing repair solution.

8. The antigen repair method using the dewaxing repair solution of claim 1, characterized in that: The following steps are involved: S1. Dewaxing and repairing: adding tissue paraffin sections into dewaxing and repairing solution, dewaxing and repairing, and drying; S2, blocking: add hydrogen peroxide blocking agent to the dried slices and incubate in the dark; S3, primary antibody incubation: add primary antibody reagent to the tissue site and incubate at a constant temperature; S4, secondary antibody incubation: add secondary antibody reagent to the tissue site and incubate at room temperature; S5, color development: add DAB working solution to the slices and incubate at room temperature in the dark; S6, counterstaining-dehydration-sealing: add hematoxylin staining solution to the slices, incubate at room temperature, dehydrate with gradient ethanol solution, dry in the air, and obtain neutral gum sealing slides; S7. Slide review: Observe the staining of the coverslips.

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