Preparation method and application of alkaline phosphatase substrate staining solution

Abstract

The invention discloses a preparation method and application of an alkaline phosphatase substrate staining solution. The preparation method comprises the following steps: S1, preparing a substrate chromogen solution and a substrate buffer solution; s2, mixing the substrate chromogen solution and the substrate buffer solution to obtain an alkaline phosphatase substrate staining solution; wherein the substrate chromogen solution comprises neofuchsin, a hydrochloric acid solution, sodium nitrite, glycerol, DMSO, a chemical stabilizer and Proclin300; the substrate buffer solution is prepared from naphthol AS-BI phosphate, a Tris buffer solution, dimethyl formamide and Tween-20. The substrate chromogen solution provided by the invention ensures the stability of the substrate chromogen, so that the alkaline phosphatase catalyzes the substrate staining solution to form a red azo precipitate insoluble in an organic solvent, the red azo precipitate can be used for immunohistochemical staining for a long time, and the prepared staining solution can be directly applied to a full-automatic immunohistochemical staining machine and has the advantages of simple preparation process and low cost. The application range of the alkaline phosphatase substrate staining solution is widened, meanwhile, the labor cost and the time cost are saved, and the efficiency and the accuracy of immunohistochemical staining are improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a preparation method of an alkaline phosphatase substrate staining solution and an application thereof. Background Art

[0002] Alkaline phosphatase (AP) is a phosphate hydrolase with broad specificity. It can catalyze all phosphatase hydrolysis reactions and phosphate group transfer reactions and has very important physiological functions in organisms. In immunohistochemistry, alkaline phosphatase (AP) and its substrates have been widely used as color development systems. Antibody-conjugated alkaline phosphatase can hydrolyze phosphates containing naphthol substitutes to produce insoluble naphthol derivatives, which then combine with suitable diazonium salts to generate colored insoluble azo dyes at the enzyme active site, ultimately causing the antigen position in the tissue section to show different colors. Commonly used substrate chromogens are BCIP / NBT (blue) and naphthol phosphate / diazo compounds (red).

[0003] At present, the most widely used immunohistochemical staining system is the peroxidase color development system, such as horseradish peroxidase (HRP). Common substrates for HRP are DAB (brown yellow) and AEC (red). However, DAB is a carcinogen, and the interpretation will be interfered by factors such as pigment deposition and red blood cells, while the precipitate formed by AEC is soluble in alcohol. Therefore, both are limited in application. The commonly used substrate for alkaline phosphatase (AP), Fast Red, is soluble in organic solvents. Therefore, it is necessary to use a water-soluble mounting medium, and the sections after color development cannot be dehydrated by ethanol and xylene, so it is not conducive to long-term storage. In addition, the dilution ratio of the chromogen and buffer of some AP substrates on the market is too high, which is not suitable for staining with a fully automatic immunohistochemical staining machine.

[0004] Therefore, there is an urgent need in the art to develop a method for preparing an alkaline phosphatase substrate staining solution that can form a precipitate insoluble in organic solvents for long-term use in immunohistochemical staining, and can also be adapted to automated equipment for staining to improve staining efficiency and save manpower and time costs. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing an alkaline phosphatase substrate staining solution, the preparation method comprising:

[0006] S1, preparing substrate chromogen solution and substrate buffer;

[0007] S2, mixing the substrate chromogen solution and the substrate buffer to obtain the alkaline phosphatase substrate staining solution;

[0008] The substrate chromogen solution includes new fuchsin, hydrochloric acid solution, sodium nitrite, glycerol, DMSO, a chemical stabilizer and Proclin300; the substrate buffer includes naphthol AS-BI phosphate, Tris buffer, dimethylformamide and Tween-20.

[0009] Specifically, in the substrate chromogen solution, the amount of new fuchsin is 0.091-0.91 g, the concentration of the hydrochloric acid solution is 1.9%, the amount of the hydrochloric acid solution is 20-50 mL, the amount of sodium nitrite is 0.052-0.52 g, the amount of glycerol is 10 mL, the amount of DMSO is 2 mL, the amount of the chemical stabilizer is 40 μL, and the amount of Proclin 300 is 10 μL. The chemical stabilizer is a Concentrated Chemical Stabilizer purchased from Biodigen Inc., which is used to maintain the stability of the reaction.

[0010] Specifically, the dosage of naphthol AS-BI phosphate is 0.3 g, the concentration of the Tris buffer is 21%, the dosage of the Tris buffer is 50 mL, the pH of the Tris buffer is 8.4, the dosage of dimethylformamide is 0.5 mL, and the dosage of Tween-20 is 0.15 mL.

[0011] Specifically, the mixing ratio of the substrate chromogen solution and the substrate buffer solution is 1:1 to 1:20.

[0012] Specifically, the mixing ratio of the substrate chromogen solution and the substrate buffer solution is 1:20.

[0013] Specifically, the substrate chromogen solution is prepared by dissolving the new fuchsin in a hydrochloric acid solution, placing the mixed solution in an ice water bath, then adding sodium nitrite to the mixed solution to obtain a suspension, and then performing solid-liquid separation on the suspension to remove the solid to obtain the substrate chromogen solution.

[0014] Specifically, the ice water bath is a 0°C ice water bath.

[0015] Specifically, the ice water bath time is 15 to 20 minutes.

[0016] Specifically, the substrate buffer solution is prepared by adding naphthol AS-BI phosphate into Tris buffer solution, adding dimethylformamide and Tween-20 into the obtained mixed solution, and mixing to obtain the substrate buffer solution.

[0017] The second aspect of the present invention provides an application of an alkaline phosphatase substrate staining solution prepared by the aforementioned preparation method of the alkaline phosphatase substrate staining solution in immunohistochemical staining, wherein the immunohistochemical staining includes manual immunohistochemical staining and immunohistochemical staining performed using a fully automatic immunohistochemical staining machine.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The preparation method provided by the present invention has a substrate chromogen solution whose formula is obtained through continuous optimization, thereby ensuring the stability of the substrate chromogen, so that the alkaline phosphatase catalyzes the substrate staining solution to form a red azo precipitate insoluble in an organic solvent, which can be used for immunohistochemical staining for a long time; at the same time, by adjusting and optimizing the use ratio of the substrate chromogen solution and the substrate buffer solution, the obtained staining solution can be directly applied to a fully automatic immunohistochemical staining machine, further improving the application scope of the alkaline phosphatase substrate staining solution, solving the manual error that may be caused by manual operation, saving labor costs and time costs, and improving the efficiency and accuracy of immunohistochemical staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a staining result diagram of a commercially available alkaline phosphatase substrate staining solution;

[0021] Figure 2 This is the staining result of alkaline phosphatase substrate staining solution 1;

[0022] Figure 3 is a staining result diagram of alkaline phosphatase substrate staining solution 2;

[0023] Figure 4 is a staining result diagram of alkaline phosphatase substrate staining solution 3;

[0024] Figure 5 is a staining result diagram of alkaline phosphatase substrate staining solution 4;

[0025] Figure 6 is a staining result diagram of alkaline phosphatase substrate staining solution 5;

[0026] Figure 7 is a staining result diagram of alkaline phosphatase substrate staining solution 6;

[0027] Figure 8 is a staining result diagram of alkaline phosphatase substrate staining solution 7;

[0028] Fig. 9 This is a staining result diagram of alkaline phosphatase substrate staining solution 4 on a fully automatic immunohistochemical staining machine. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the embodiments, unless otherwise specified, are conventional means well known to those skilled in the art.

[0030] Example 1 Preparation of alkaline phosphatase substrate staining solution

[0031] This example shows the preparation process of an alkaline phosphatase substrate staining solution obtained by mixing substrate chromogen solutions under two formulations with substrate buffer solutions at different ratios.

[0032] In the preparation of substrate chromogen solution, this application shows two formulations:

[0033] Formula 1: Dissolve 0.091 g of new fuchsin in 20 ml of 1.9% hydrochloric acid solution, place the solution in a 0°C ice water bath, and slowly add 0.052 g of sodium nitrite over 15 minutes to obtain a suspension, separate the suspension into solid and liquid, remove the solid, and obtain a substrate chromogen formula 1 solution;

[0034] Formula 2: Dissolve 0.91 g of new fuchsin in 50 ml of 1.9% hydrochloric acid solution, place the solution in a 0°C ice water bath, and slowly add 0.52 g of sodium nitrite over 20 minutes to obtain a suspension, separate the suspension into solid and liquid, remove the solid, and obtain a substrate chromogen formula 2 solution;

[0035] Preparation of substrate buffer: 0.3 g of naphthol AS-BI phosphate was added to 50 ml of 21% Tris buffer with a pH of 8.4, and the mixed solution was mixed with 0.5 ml of dimethylformamide and 0.15 ml of Tween-20 to obtain a substrate buffer.

[0036] Preparation of alkaline phosphatase substrate staining solution:

[0037] Alkaline phosphatase substrate staining solution 1: mix the substrate color source formula 1 solution and the substrate buffer solution at a ratio of 1:1 to obtain alkaline phosphatase substrate staining solution 1;

[0038] Alkaline phosphatase substrate staining solution 2: mix the substrate chromogen formula 1 solution with the substrate buffer at a ratio of 1:10 to obtain alkaline phosphatase substrate staining solution 2;

[0039] Alkaline phosphatase substrate staining solution 3: Mix the substrate color source formula 2 solution and the substrate buffer solution at a ratio of 1:20 to obtain alkaline phosphatase substrate staining solution 3.

[0040] The prepared alkaline phosphatase substrate staining solution was stored away from light for future use.

[0041] In the immunohistochemical reaction, the alkaline phosphatase carried by the antibody reacts with naphthol AS-BI phosphate in the substrate buffer, and the reacted substance reacts with the substrate chromogen (mainly diazonium salt), ultimately causing the antigenic site in the tissue section to appear red.

[0042] Example 2 Comparison of the effects of the alkaline phosphatase substrate staining solution obtained by the preparation method provided by this application and the prior art

[0043] The three alkaline phosphatase substrate staining solutions prepared in Example 1 were combined with commercially available alkaline phosphatase substrate staining solutions, mouse anti-p16 antibody and alkaline phosphatase-labeled goat anti-mouse secondary antibody to perform immunohistochemical staining of cervical cancer tissues.

[0044] The test steps are as follows:

[0045] 1) Sample preparation: Formalin-fixed paraffin-embedded sections were baked in a 60°C incubator for 1-2 hours and stored at room temperature for later use.

[0046] 2) Dewaxing and hydration:

[0047] Paraffin sections were placed in fresh xylene and immersed for 10 min × 2 times;

[0048] After removing excess liquid, place in anhydrous ethanol and soak for 3 minutes × 2 times;

[0049] After removing excess liquid, place in 95% ethanol and soak for 3 minutes;

[0050] After removing excess liquid, place in 85% ethanol and soak for 3 minutes;

[0051] After removing excess liquid, place in 70% ethanol and soak for 3 minutes;

[0052] Rinse with purified water for 3 minutes × 2 times.

[0053] 3) Antigen repair: Use pH 9.0 EDTA high temperature heat repair method, heat repair in a pressure cooker for 5 minutes, let the slices cool naturally to room temperature. Rinse with purified water twice, 3 minutes each time, and circle the tissue to be tested with an immunohistochemistry pen.

[0054] 4) Inactivation of endogenous peroxidase: drop an appropriate amount of endogenous peroxidase blocker containing 3% hydrogen peroxide until the tissue is completely covered, incubate at room temperature for 5 to 15 minutes, rinse twice with purified water for 3 minutes each time, and rinse with PBS for 3 minutes.

[0055] 5) Primary antibody incubation: Add 1 to 3 drops of p16 antibody reagent to completely cover the tissue, incubate at room temperature for 60 minutes, and then rinse with PBS three times, 5 minutes each time.

[0056] 6) Secondary antibody incubation: Add 1 to 3 drops of alkaline phosphatase-labeled goat anti-mouse secondary antibody, incubate at room temperature for 30 minutes, and then rinse with PBS three times, 5 minutes each time.

[0057] 7) AP color development: add 100-150 μL alkaline phosphatase substrate staining solution, color development for 8 minutes, and rinse with PBS 3 times, 3 minutes each time.

[0058] 8) Hematoxylin staining: Add 100-150 μL of hematoxylin staining solution to the slices for 2 minutes, then rinse with PBS or tap water to turn blue. 9) Dehydration and transparency: Soak in gradient alcohol 70%, 85%, 95%, 100%, 100% in sequence, 3 minutes each time; transparentize twice in xylene, 10 minutes each time. (This step is omitted for tissue slices using commercially available alkaline phosphatase substrate staining solution)

[0059] 10) Sealing: Seal the samples with neutral gum.

[0060] The tissue sections stained with commercially available alkaline phosphatase substrate solution were not subjected to the dehydration and clearing step 9). The specific staining results are shown in Figure 1 .

[0061] The tissue sections using the three alkaline phosphatase substrate staining solutions prepared in Example 1 of the present invention can be dehydrated and made transparent. The staining results of alkaline phosphatase substrate staining solution 1 and alkaline phosphatase substrate staining solution 2 are shown in FIG. Figure 2 and Figure 3 , the staining results have some background, the staining results of alkaline phosphatase substrate staining solution 3 are shown in Figure 4 It can be seen that the red staining site positioning is clearer than the staining results of the existing commercial technology, and there is no halo or fading. Therefore, alkaline phosphatase substrate staining solution 3 is preferred, that is, the substrate color original formula 2 solution and the substrate buffer solution are mixed at a ratio of 1:20.

[0062] Example 3 Study on the Stability of the Substrate Chromogen Solution

[0063] This example shows the process of stability research of substrate chromogen solution during the research and development process. On the basis of substrate chromogen solution formula 2 in Example 1, substrate chromogen formula 3 and substrate chromogen formula 4 are prepared, and on the basis of them, alkaline phosphatase substrate staining solution 4, alkaline phosphatase substrate staining solution 5, alkaline phosphatase substrate staining solution 6 and alkaline phosphatase substrate staining solution 7 are prepared. The specific process is as follows:

[0064] Substrate chromogen preparation:

[0065] Formula 3: Take 20 ml of the substrate chromogen of Formula 2, add 10 ml of glycerol, 2 ml of DMSO, 40 ul of a chemical stabilizer and 10 ul of Proclin300 to obtain the substrate chromogen formula 3 solution;

[0066] Formula 4: Take 20 ml of the substrate chromogen of Formula 2, add 10 ml of glycerol, 2 ml of DMSO, 0.2 g of bovine serum albumin, 0.002 g of casein, 0.36 g of polyethylene glycol 2000, 0.02 g of polyvinyl pyrrolidone K30, 40 ul of chemical stabilizer and 10 ul of Proclin300 to obtain the substrate chromogen formula 4 solution;

[0067] The substrate color original formula 3 solution and the substrate color original formula 4 solution were accelerated to stand for 11 days.

[0068] The preparation of substrate buffer is the same as that in Example 1.

[0069] Preparation of substrate staining solution:

[0070] Alkaline phosphatase substrate staining solution 4: mix the substrate chromogen formula 3 solution with the substrate buffer solution at a ratio of 1:20 to obtain alkaline phosphatase substrate staining solution 4;

[0071] Alkaline phosphatase substrate staining solution 5: Mix the accelerated 11-day substrate chromogen formula 3 solution with the substrate buffer at a ratio of 1:20 to obtain alkaline phosphatase substrate staining solution 5;

[0072] Alkaline phosphatase substrate staining solution 6: mix the substrate chromogen formula 4 solution with the substrate buffer at a ratio of 1:20 to obtain alkaline phosphatase substrate staining solution 6;

[0073] Alkaline phosphatase substrate staining solution 7: Mix the accelerated 11-day substrate chromogen formula 4 solution with the substrate buffer at a ratio of 1:20 to obtain alkaline phosphatase substrate staining solution 7;

[0074] The prepared alkaline phosphatase substrate staining solution was stored away from light for future use.

[0075] The above four alkaline phosphatase substrate staining solutions were combined with p16 / Ki-67 mixed antibodies to perform manual immunohistochemical staining of cervical cancer tissues, and the steps were as follows:

[0076] 1. Sample preparation: Bake the formalin-fixed paraffin-embedded sections in a 60°C incubator for 1 to 2 hours and store them at room temperature for later use.

[0077] 2. Dewaxing and hydration:

[0078] Paraffin sections were placed in fresh xylene and immersed for 10 min × 2 times;

[0079] After removing excess liquid, place in anhydrous ethanol and soak for 3 minutes × 2 times;

[0080] After removing excess liquid, place in 95% ethanol and soak for 3 minutes;

[0081] After removing excess liquid, place in 85% ethanol and soak for 3 minutes;

[0082] After removing excess liquid, place in 70% ethanol and soak for 3 minutes;

[0083] Rinse with purified water for 3 minutes × 2 times.

[0084] 3. Antigen repair: It is recommended to use pH 9.0 EDTA high temperature heat repair method, heat repair in a pressure cooker for 5 minutes, and let the slices cool naturally to room temperature. Rinse with purified water twice, 3 minutes each time, and circle the tissue to be tested with an immunohistochemical pen.

[0085] 4. Inactivation of endogenous peroxidase: drop an appropriate amount of endogenous peroxidase blocker containing 3% hydrogen peroxide to completely cover the tissue. Incubate at room temperature for 5 to 15 minutes, then rinse twice with purified water for 3 minutes each time, and then rinse with PBS for 3 minutes.

[0086] 5. Primary antibody incubation: Add 1 to 3 drops of p16 / Ki-67 combination primary antibody to completely cover the tissue, incubate at room temperature for 60 minutes, and rinse with PBS three times, 5 minutes each time.

[0087] 6. Secondary antibody incubation: Add 1 to 3 drops of AP / HRP enzyme-labeled secondary antibody, incubate at room temperature for 30 minutes, and then rinse with PBS three times, 5 minutes each time.

[0088] 7. AP color development: add 100-150ul of different alkaline phosphatase staining solutions, color development for 8 minutes, and rinse with PBS 3 times, 3 minutes each time.

[0089] 8. DAB color development: Add 100-150ul of prepared DAB staining solution and color develop for 5 minutes. The staining can be terminated according to the color change. Rinse with purified water twice, 3 minutes each time.

[0090] 9. Hematoxylin staining: Add 100-150ul of hematoxylin staining solution to the sections for 2 minutes, then rinse with PBS or tap water to turn blue.

[0091] 10. Dehydration and transparency: Soak in gradient alcohol 70%, 85%, 95%, 100%, 100% in sequence, 3 minutes each time; transparentize in xylene twice, 10 minutes each time.

[0092] 11. Seal the slides: Seal the samples with neutral gum.

[0093] like Figure 5 , Figure 6 and Figure 7 As shown, they correspond to cervical cancer tissue sections using alkaline phosphatase substrate staining solution 4, alkaline phosphatase substrate staining solution 5 and alkaline phosphatase substrate staining solution 6 in Example 3 of the present invention, wherein the red staining site is clearly located, and the cytoplasm of the same cell in the tissue is red-colored, the cell nucleus is dark brown or reddish brown, and the cytoplasm does not appear halo or fading; Figure 8 As shown, the cervical cancer tissue section was treated with the alkaline phosphatase substrate staining solution 8 in Example 3 of the present invention, wherein the staining intensity of the red staining area was significantly weakened. Therefore, the stability of the substrate chromogen formula 3 is better.

[0094] Example 4: Fully automatic immunohistochemical staining machine for immunohistochemical staining test

[0095] This example shows the process and results of immunohistochemical staining of cervical cancer tissue using the alkaline phosphatase substrate staining solution 4 prepared in Example 3 in combination with the p16 / Ki-67 mixed antibody on a fully automatic immunohistochemical staining machine.

[0096] 1. Sample preparation: Bake the formalin-fixed paraffin-embedded sections in a 60°C incubator for 1 to 2 hours and store them at room temperature for later use.

[0097] 2. The reaction conditions of the fully automatic immunohistochemical staining machine are as shown in Table 1.

[0098] Table 1

[0099] Serial number step parameter 1 Antigen retrieval High temperature 100℃ repair for 30min 2 Peroxidase <![CDATA[Treatment with 3% H2O2 for 10 min]]> 3 p16 / Ki-67 mixed primary antibody Incubate at room temperature for 30 min 4 AP+HRP enzyme-labeled secondary antibody Incubate at room temperature for 30 min 5 AP color development 8min 6 DAB color development 5min 7 Hematoxylin 3min

[0100] 3. Dehydration and transparentization: After the staining process is completed, remove the slices and soak them in gradient alcohol 70%, 85%, 95%, 100%, 100% in sequence for 3 minutes each time; and transparentize them in xylene twice for 10 minutes each time.

[0101] 4. Seal the slides: Seal the samples with neutral gum.

[0102] like Fig. 9As shown, this is the staining result of alkaline phosphatase substrate staining solution 4 on a fully automatic immunohistochemical staining machine. It can be seen that the red staining site of the cervical cancer tissue section is clearly located, and the cytoplasm of the same cell in the tissue is red and the nucleus is dark brown or reddish brown, and there is no halo or fading in the cytoplasm.

[0103] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an alkaline phosphatase substrate staining solution, characterized in that: The preparation method comprises: S1, preparing substrate chromogen solution and substrate buffer; S2, mixing the substrate chromogen solution and the substrate buffer to obtain the alkaline phosphatase substrate staining solution; The substrate chromogen solution includes new fuchsin, hydrochloric acid solution, sodium nitrite, glycerol, DMSO, a chemical stabilizer and Proclin300; the substrate buffer includes naphthol AS-BI phosphate, Tris buffer, dimethylformamide and Tween-20.

2. The method for preparing an alkaline phosphatase substrate staining solution according to claim 1, characterized in that: In the substrate chromogen solution, the dosage of new fuchsin is 0.091-0.91 g, the concentration of the hydrochloric acid solution is 1.9%, the dosage of the hydrochloric acid solution is 20-50 mL, the dosage of sodium nitrite is 0.052-0.52 g, the dosage of glycerol is 10 mL, the dosage of DMSO is 2 mL, the dosage of the chemical stabilizer is 40 μL, and the dosage of Proclin300 is 10 μL.

3. The method for preparing an alkaline phosphatase substrate staining solution according to claim 1, characterized in that: The dosage of the naphthol AS-BI phosphate is 0.3 g, the concentration of the Tris buffer is 21%, the dosage of the Tris buffer is 50 mL, the pH of the Tris buffer is 8.4, the dosage of the dimethylformamide is 0.5 mL, and the dosage of the Tween-20 is 0.15 mL.

4. The method for preparing an alkaline phosphatase substrate staining solution according to claim 1, characterized in that: The mixing ratio of the substrate chromogen solution and the substrate buffer solution is 1:1 to 1:

20.

5. The method for preparing an alkaline phosphatase substrate staining solution according to claim 1, characterized in that: The mixing ratio of the substrate chromogen solution and the substrate buffer solution is 1:

20.

6. The method for preparing an alkaline phosphatase substrate staining solution according to claim 1, characterized in that: The substrate chromogen solution is prepared by dissolving the new fuchsin in a hydrochloric acid solution, placing the mixed solution in an ice water bath, then adding sodium nitrite to the mixed solution to obtain a suspension, and performing solid-liquid separation on the suspension to remove solids to obtain the substrate chromogen solution.

7. The method for preparing an alkaline phosphatase substrate staining solution according to claim 6, characterized in that: The ice water bath is a 0°C ice water bath.

8. The method for preparing an alkaline phosphatase substrate staining solution according to claim 6, characterized in that: The ice water bath time is 15 to 20 minutes.

9. The method for preparing an alkaline phosphatase substrate staining solution according to claim 1, wherein the substrate buffer is prepared by adding naphthol AS-BI phosphate to a Tris buffer, adding dimethylformamide and Tween-20 to the resulting mixed solution, and mixing to obtain a substrate buffer.

10. Use of the alkaline phosphatase substrate staining solution prepared by the preparation method of the alkaline phosphatase substrate staining solution according to any one of claims 1 to 9 in immunohistochemical staining, characterized in that: The immunohistochemical staining includes manual immunohistochemical staining and immunohistochemical staining performed using a fully automatic immunohistochemical staining machine.

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