DAB buffer and DAB staining liquid kit for DAB coloration

Abstract

The application relates to the field of immunohistochemical staining, in particular to a DAB buffer and a DAB staining solution kit for DAB color development. The application discloses a DAB buffer for DAB color development, and the main components are hydrogen peroxide, nonylphenol polyoxyethylene ether, magnesium sulfate and trisodium citrate. The metal ions in the DAB buffer can enhance the electron transfer speed between DAB and hydrogen peroxide, and improve the color development sensitivity. The nonylphenol polyoxyethylene ether can stabilize the hydrogen peroxide, so that the hydrogen peroxide can be stored for a longer time and the cost is saved. The application further discloses a DAB staining solution kit, which comprises a blocking agent, a reaction enhancer, an enzyme-labeled polymer IgG, a DAB concentrated solution, a DAB buffer and hematoxylin, and the above-mentioned DAB buffer for DAB color development. The DAB staining solution kit of the application can improve the detection sensitivity of immunohistochemical staining, and can also improve the storage stability of the DAB color development solution and reduce the cost.

Description

Technical Field

[0001] This application relates to the field of immunohistochemical staining, specifically to DAB buffer and DAB staining kits for DAB staining. Background Technology

[0002] Existing DAB staining kits generally contain components such as blocking agents, reaction enhancement solutions, enzyme-labeled polymer IgG, DAB concentrate, DAB buffer, and hematoxylin, and are mainly used for immunohistochemical staining in pathology departments. Immunohistochemical staining is one of the commonly used techniques in pathology departments. It utilizes the immunological principle of specific binding between antigens and antibodies, and through an enzymatic reaction, causes the antigen-antibody-enzyme-labeled polymer IgG complex to develop color in situ, thereby determining whether the target antigen is present in tissue cells and performing localization and qualitative detection of the target antigen.

[0003] In DAB staining kits, enzyme-labeled polymeric IgG (commonly known as secondary antibody or polymeric secondary antibody) is the most important component. Traditional secondary antibodies typically involve goat anti-mouse or goat anti-rabbit antibodies binding to peroxidase (HRP) under the action of a compound. This method suffers from low binding efficiency and poor detection sensitivity, failing to meet the needs of in vitro diagnostic reagents. Most recent polymeric secondary antibodies use polymers as substrates, coupling goat anti-mouse or goat anti-rabbit antibodies and HRP enzymes to the polymer through its extensive binding sites. Using polymers as substrates allows for the binding of large amounts of antibodies and HRP enzymes, thus significantly improving detection sensitivity and staining efficacy compared to traditional secondary antibodies. However, due to differences in polymeric secondary antibody raw material technology and processes, the staining quality varies considerably between different polymeric secondary antibody raw materials, and the raw material prices are generally high.

[0004] Improving the colorimetric intensity is crucial when using DAB staining kits. Current technology typically uses 3,3-diaminobenzidine (DAB) as the substrate, with HRP-labeled polymeric IgG catalyzing the DAB staining process. The DAB staining solution is generally prepared by mixing DAB concentrate and DAB buffer at a 1:20 ratio. The DAB buffer contains hydrogen peroxide (H2O2), which is essential for colorimetric development. However, due to the instability and easy decomposition of H2O2, the DAB buffer often becomes ineffective due to H2O2 decomposition, resulting in pale or negative immunohistochemical staining. Therefore, maintaining the stability of H2O2 in the DAB buffer is critical.

[0005] The colorimetric principle of DAB is as follows: HRP catalyzes the oxidation of DAB by H2O2 to form reduced DAB. Reduced DAB is insoluble in water and forms a brownish-yellow precipitate in situ within the antigen-antibody-enzyme-labeled polymer IgG complex. During this reaction, due to the high standard potential difference between H2O2 and DAB, and the inherent instability of H2O2's physicochemical properties, the prepared DAB colorimetric solution will gradually oxidize and become ineffective, and cannot be stored for extended periods. Therefore, existing kits use DAB concentrate and H2O2 buffer as colorimetric reagents, prepared and stored separately, and prepared fresh at a 1:20 ratio before use.

[0006] In summary, the main problems with DAB staining kits include: ① low kit sensitivity due to the instability of H2O2; ② poor stability and short shelf life of the prepared DAB chromogenic solution. Currently, Chinese invention patent CN113484313A improves the intensity and sensitivity of immunohistochemical detection by adding a DAB enhancer, comprising copper sulfate, sodium oxide, potassium oxide, sucrose, and benzalkonium chloride; protects and stabilizes hydrogen peroxide using sodium pyrophosphate and EDTA; and increases stability by delaying the oxidation rate of DAB using methyl ethylene glycol. Chinese invention patent CN1595150A improves stability by adding carbohydrates and polymers to inhibit the auto-oxidation activity of the chromogenic substance. Chinese invention patent CN110887831A addresses the problems of poor stability and short shelf life of DAB chromogenic solutions by providing aminoglucan. Chinese invention patent CN110361245A increases stability by adding disodium dihydrogen pyrophosphate to the buffer solution to protect hydrogen peroxide. Chinese invention patent CN110567785A improves the stability of DAB working solutions by adding epigallocatechin gallate or its derivatives.

[0007] However, the aforementioned patents cannot improve the detection sensitivity of immunohistochemical staining while also improving the preservation stability of DAB chromogenic solution and reducing costs, so further improvements are needed. Summary of the Invention

[0008] In order to improve the detection sensitivity of immunohistochemical staining, while also improving the storage stability of DAB chromogenic solution and reducing costs, this application provides a DAB buffer and DAB staining solution kit for DAB chromogenic staining.

[0009] In a first aspect, this application discloses a DAB buffer for DAB color development, wherein the main components of the DAB buffer are hydrogen peroxide, nonylphenol polyoxyethylene ether, magnesium sulfate and trisodium citrate.

[0010] By employing the above technical solution, the magnesium sulfate and trisodium citrate in the DAB buffer contain metal ions, which enhance the electron transfer rate between DAB and hydrogen peroxide. This results in a shorter reaction time and higher colorimetric efficiency for the DAB chromogenic solution, ultimately improving the sensitivity of DAB colorimetry and consequently enhancing the detection sensitivity of the DAB staining kit. Nonylphenol polyoxyethylene ether stabilizes hydrogen peroxide, preventing its decomposition and allowing for longer storage after preparation, thus saving costs compared to freshly prepared DAB buffers.

[0011] In some embodiments, the DAB buffer is a mixture of hydrogen peroxide, imidazole, nonylphenol polyoxyethylene ether, chloroalkyl dimethylbenzylamine, magnesium sulfate, and trisodium citrate.

[0012] By adopting the above technical solution, magnesium sulfate and trisodium citrate in the DAB buffer enhance the electron transfer rate between DAB and hydrogen peroxide, resulting in a shorter reaction time and higher colorimetric efficiency of the DAB chromogenic solution, ultimately improving the sensitivity of DAB colorimetry and thus enhancing the detection sensitivity of the DAB staining kit. Furthermore, imidazole, nonylphenol polyoxyethylene ether, and chloroalkyl dimethylbenzylamine in the DAB buffer more effectively stabilize hydrogen peroxide. Therefore, after mixing the DAB buffer with the DAB concentrate in the correct proportion, low-temperature storage has minimal impact on its effectiveness, improving the storage stability of the DAB chromogenic solution. It still maintains good staining effects after several days, thus reducing the frequency of preparation by experimenters and allowing for more flexible experimental arrangements. Moreover, the DAB buffer disclosed in this application is relatively simple to prepare and can be used multiple times within a few days without waste. Compared to existing commercially available DAB buffers that are prepared and used immediately and quickly expire, this solution effectively reduces costs due to its lower market price and higher utilization rate.

[0013] In some embodiments, the hydrogen peroxide has a volume concentration of 0.05-0.3%, the imidazole has a molar concentration of 30-80 mM, the nonylphenol polyoxyethylene ether has a mass percentage concentration of 0.04-0.18%, the chloroalkyl dimethylbenzylamine has a volume concentration of 0.006-0.015%, the magnesium sulfate has a molar concentration of 0.5-2.2 mM, and the trisodium citrate has a molar concentration of 0.6-1.8 mM.

[0014] By adopting the above technical solution, the DAB buffer obtained after mixing various components at the above concentrations can better slow down the decomposition rate of hydrogen peroxide, improve the stability of the DAB buffer, thereby further improving the sensitivity of the kit, and allowing the DAB chromogenic solution to be stored for a longer time after preparation.

[0015] Secondly, this application discloses a DAB staining reagent kit, comprising an inhibitor, a reaction enhancement solution, an enzyme-labeled polymer IgG, a DAB concentrate, a DAB buffer, and hematoxylin, wherein the DAB buffer is the DAB buffer described above for DAB staining.

[0016] In some embodiments, the blocking agent is a hydrogen peroxide solution with a concentration of 1-6%.

[0017] In some embodiments, the main component of the reaction-enhancing solution is rabbit anti-mouse immunoglobulin.

[0018] In some embodiments, the concentration of the reaction enhancement solution is 130-550 ug / mL.

[0019] In some embodiments, the DAB concentrate is prepared from 1,2-propanediol, water, and DAB.

[0020] By employing the above technical solution, a 1%-6% hydrogen peroxide solution can inactivate endogenous peroxidase within a certain time. Rabbit anti-mouse immunoglobulin can further promote binding, making the colorimetric reaction more sensitive. 1,2-Propanediol in the DAB concentrate can inhibit the spontaneous oxidation of DAB, thus extending the usable time after preparation.

[0021] In some embodiments, the enzyme-labeled polymer IgG is obtained by the following preparation method:

[0022] S1. Mix 3-15 mg / mL of goat anti-rabbit antibody and 2-10 mg / mL of goat anti-mouse antibody evenly, then add 12-26 mg / mL of ethylene glycol chitosan polymer and mix evenly. Then add 20-50 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3-8 mg / mL of N-hydroxysulfosuccinimide. Mix well and adjust the pH to 2.2-5.4 with 0.03-0.2 M HCl. React at room temperature in the dark for 2-8 hours to obtain solution A for later use.

[0023] S2. Add 6-16 mg / ml of peroxidase solution to 18-30 mg / ml of polyethylene glycol 2000 solution, then add 2-8 mg / ml of polyethylene glycol-modified SMCC crosslinking agent, react in the dark for 2-8 hours to obtain solution B for later use.

[0024] S3. Mix the above solutions A and B, add 1-4 mg / ml of polyethylene glycol-modified SMCC crosslinking agent, adjust the pH to 6.5-7.5, react in the dark for 2-6 hours, and obtain the product after adsorption and elution.

[0025] By employing the above-mentioned technical solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) can activate the coupling of carboxyl and amine groups to form amide bonds, while N-hydroxysulfosuccinimide (NHS) can improve the efficiency of the EDAC coupling reaction. NHS can control and modify the carbodiimide crosslinking reaction and is used for the activation of carboxylates (-COOH) coupled with primary amines (-NH2). Derivatives can be synthesized by mixing sulfonyl-NHS with carboxyl-containing molecules and carbodiimide (EDAC).

[0026] PEGylated SMCC crosslinking agents can react with amino and thiol groups to irreversibly crosslink proteins or peptides through flexible PEG spacer arms. Therefore, SMCC crosslinking agents can couple polyethylene glycol 2000 molecules to peroxidase molecules, and the polyethylene glycol spacer arms help maintain the solubility of the conjugate.

[0027] After mixing solutions A and B, the SMCC cross-linking agent cross-links between primary amines (-NH2) and thiol groups (-SH) in proteins and other molecules. The NHS groups react specifically and efficiently with lysine and N-terminal amino groups in peroxidase molecules at pH 7-9, forming stable amide bonds. Maleimide groups react with reduced thiol groups at pH 6.5-7.5, forming stable thioether bonds. After purification, the above mixture yields the enzyme-labeled polymer IgG.

[0028] The enzyme-labeled polymer IgG prepared by this method has the characteristics of high reaction sensitivity, universal applicability to mice and rabbits, no background and non-specific staining, and can be used as the secondary antibody in this DAB kit, which greatly improves the sensitivity and colorimetric properties of the kit.

[0029] Thirdly, this application discloses a method for applying a DAB staining reagent kit, the method of applying the DAB staining reagent kit comprising the following steps:

[0030] A1: Dewaxing:

[0031] a1. Bake the cut tissue chips at 60-70℃ for 25-35 minutes;

[0032] a2. After baking, directly immerse in xylene for dewaxing three times, each time for 4-6 minutes;

[0033] A2: Hydration:

[0034] b1. After dewaxing, the sections were hydrated twice with 100% alcohol for 2-4 minutes each time, then hydrated with 95% alcohol for 2-4 minutes each time, and then hydrated with 85% alcohol for 2-4 minutes each time.

[0035] b2. Then soak the hydrated slices in purified water for 1-3 minutes;

[0036] A3: Antigen thermal retrieval:

[0037] c1. Add EDTA repair solution with pH 9.0, heat until the EDTA repair solution begins to boil, then add the hydrated slices, and then let the EDTA repair solution boil intermittently for 20 minutes, and then slowly cool to room temperature.

[0038] c2. Soak the repaired slices in distilled water for 1-3 minutes;

[0039] A4: Blocking endogenous peroxidase:

[0040] d1. Remove the liquid from the slide and delineate the area of ​​tissue to be tested on the slide;

[0041] d2. Then wash the slides with PBST 4 times, 1-3 minutes each time, taking care to prevent the slides from drying out;

[0042] d3. Evenly drop 90-110 uL of blocking agent onto each tissue section and incubate at room temperature for 8-12 min;

[0043] d4. Wash off the blocking agent and rinse 4 times with PBST solution, 1-3 min each time;

[0044] A5: Primary antibody incubation:

[0045] e1. Discard the PBST solution, add 100 μL of primary antibody working solution evenly to each slide, and incubate at room temperature for 28-32 min;

[0046] e2. After incubation, rinse 4 times with PBST solution, 1-3 minutes each time;

[0047] A6: Incubation with reaction-enhancing solution:

[0048] f1. Discard the PBST solution, and evenly add 100 μL of reaction enhancement solution to each slide. Incubate at room temperature for 10-20 min.

[0049] f2. After incubation, rinse 4 times with PBST solution, 1-3 min each time;

[0050] A7: Secondary antibody incubation:

[0051] g1. Discard the PBST solution, and evenly add 100 μL of enzyme-labeled polymer IgG to each slice. Incubate at room temperature for 10-20 min.

[0052] g2. After incubation, rinse 4 times with PBST solution, 1-3 min each time;

[0053] A8: DAB color rendering:

[0054] h1. Discard the PBST solution, and evenly add 150 μL of freshly prepared DAB chromogenic solution using DAB buffer and DAB concentrate to each slide. Incubate at room temperature for 4-6 min.

[0055] h2. Observe the staining results, then rinse with tap water for 2-4 minutes;

[0056] A9: Hematoxylin counterstaining:

[0057] j1. Remove the water from the tissue, then add 150 μL of hematoxylin staining solution for counterstaining and incubate for 1-3 min;

[0058] j2. Then immediately rinse it in tap water and soak it in tap water for 2-4 minutes to restore its blue color;

[0059] A10: Dehydration sealing sheet:

[0060] k1. After the slices turn blue, shake off the water, and then dehydrate them in 85% alcohol for 1-3 minutes, 95% alcohol for 1-3 minutes, and 100% alcohol twice for 1-3 minutes each time.

[0061] k2. After removing the sections, dry them at 60-70℃ for 8-12 minutes. After drying, add neutral resin to the sections and mount them.

[0062] By adopting the above technical solution, the DAB staining reagent kit disclosed in this application, using the above application method, can maintain good stability during use, is not easily affected by changes in color development sensitivity, and can achieve extremely high staining quality.

[0063] In summary, this application includes at least one of the following beneficial technical effects:

[0064] 1. The magnesium sulfate and trisodium citrate in the DAB buffer disclosed in this application enhance the electron transfer rate between DAB and hydrogen peroxide, resulting in a shorter reaction time and higher colorimetric efficiency of the DAB chromogenic solution, ultimately improving the sensitivity of DAB colorimetry and thus enhancing the detection sensitivity of the DAB staining kit. Furthermore, the nonylphenol polyoxyethylene ether and other components in the DAB buffer stabilize hydrogen peroxide; therefore, after mixing the DAB buffer with DAB concentrate in the correct proportion, low-temperature storage has minimal impact on its effectiveness, improving the storage stability of the DAB chromogenic solution. It still maintains good staining effects after several days, thus reducing the frequency of preparation by experimenters and allowing for more flexible experimental scheduling. Moreover, the DAB buffer disclosed in this application is simple to prepare and can be used multiple times within a few days without waste. Compared to existing commercially available DAB buffers that are prepared and used immediately and quickly expire, it effectively reduces costs due to both its lower market price and higher utilization rate.

[0065] 2. This application discloses a method for preparing enzyme-labeled polymer IgG. The enzyme-labeled polymer IgG prepared by this method has the characteristics of high sensitivity and good specificity. When used in combination with the disclosed DAB buffer and DAB concentrate, it can achieve extremely high staining quality. Moreover, the market price is low, and it has good application prospects. Attached Figure Description

[0066] Figure 1 These are the staining results of Example 1(a) and Comparative Example 7(b) in this application;

[0067] Figure 2 These are the staining results of Example 3(a) and Comparative Example 3(b) of this application;

[0068] Figure 3 These are the staining results of Example 4(a) and Comparative Example 4(b) of this application;

[0069] Figure 4 These are the staining results of Example 5(a) and Comparative Example 5(b) of this application;

[0070] Figure 5 These are the staining results of Example 6(a) and Comparative Example 6(b) of this application;

[0071] Figure 6 The staining results are obtained by placing the prepared DAB chromogenic solution at 2-8℃ for 0 days.

[0072] Figure 7 The staining results are obtained by placing the prepared DAB chromogenic solution at 2-8℃ for 3 days.

[0073] Figure 8The staining results are obtained by placing the prepared DAB colorimetric solution at 2-8℃ for 7 days.

[0074] Figure 9 The results are the color development of the DAB colorimetric solution (a) of this application and the old DAB colorimetric solution (b) after 10 seconds of preparation. Detailed Implementation

[0075] The present application will be further described in detail below with reference to the accompanying drawings, preparation examples, embodiments, and comparative examples. All reagents used in this application, except those prepared in this application, are commercially available.

[0076] Preparation Example 1

[0077] Preparation of DAB staining solution kit:

[0078] 1. The preparation method of DAB buffer is as follows: Add 3 mL of hydrogen peroxide with a volume concentration of 0.3%, 3.4 g of imidazole with a molar concentration of 50 mM, 1 g of nonylphenol polyoxyethylene ether with a mass concentration of 0.1%, 100 μL of chloroalkyl dimethylbenzylamine with a volume concentration of 0.01%, 0.12 g of magnesium sulfate with a molar concentration of 1 mM, and 0.258 g of trisodium citrate with a molar concentration of 1 mM to 1000 mL of purified water, and mix well.

[0079] 2. Preparation of the blocking agent: The blocking agent is a 3% hydrogen peroxide solution.

[0080] 3. Preparation of reaction enhancement solution: Commercially available, diluted to 320ug / mL using antibody diluent.

[0081] 4. Preparation of enzyme-labeled polymer IgG:

[0082] S1. Mix 100 mL of 9 mg / mL goat anti-rabbit antibody and 100 mL of 6 mg / mL goat anti-mouse antibody evenly, then add 100 mL of 18 mg / mL ethylene glycol chitosan polymer and mix evenly. Then add 20 mL of 40 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 60 mL of 5 mg / mL N-hydroxysulfosuccinimide. Mix well and adjust the pH to 3.0 with 0.1 M HCl. React at room temperature in the dark for 6 hours to obtain solution A for later use.

[0083] S2. Add 200 mL of 12 mg / mL peroxidase solution to 140 mL of 25 mg / mL polyethylene glycol 2000 solution, then add 250 mL of 4 mg / mL polyethylene glycol-modified SMCC crosslinking agent, react in the dark for 4 hours, and obtain solution B for later use.

[0084] S3. Mix the above solutions A and B, add 800 mL of 2 mg / mL polyethylene glycol-modified SMCC crosslinking agent, adjust the pH to 7.3, react in the dark for 4 hours, elute the resulting mixture by ion exchange column chromatography, and then elute again with protein A. Finally, dilute the eluted antibody with diluent and perform quality inspection.

[0085] 5. Preparation of DAB concentrate: Mix 20% 1,2-propanediol and 80% water, then add DAB to prepare a solution with a DAB mass-volume ratio of 2%.

[0086] 6. Preparation of hematoxylin: 1.2g hematoxylin, 50g potassium aluminum sulfate, 0.2g sodium iodate, 50g chloral hydrate and 20ml glacial acetic acid were added to 1000mL of water to prepare hematoxylin.

[0087] Preparation Example 2

[0088] The difference between Preparation Example 2 and Preparation Example 1 is that the DAB buffer was prepared by adding 3 mL of hydrogen peroxide with a volume concentration of 0.3%, 1 g of nonylphenol polyoxyethylene ether with a mass concentration of 0.1%, 0.12 g of magnesium sulfate with a molar concentration of 1 mM and 0.258 g of trisodium citrate with a molar concentration of 1 mM to 1000 mL of purified water and mixing them thoroughly.

[0089] Preparation Example 3

[0090] The difference between Preparation Example 3 and Preparation Example 1 is that the DAB buffer was prepared by adding 3 mL of hydrogen peroxide with a volume concentration of 0.3%, 0.12 g of magnesium sulfate with a molar concentration of 1 mM and 0.258 g of trisodium citrate with a molar concentration of 1 mM to 1000 mL of purified water and mixing them thoroughly.

[0091] Preparation Example 4

[0092] The difference between Preparation Example 4 and Preparation Example 1 is that the DAB buffer was prepared by adding 3 mL of hydrogen peroxide with a volume concentration of 0.3% and 1 g of nonylphenol polyoxyethylene ether with a mass concentration of 0.1% to 1000 mL of purified water.

[0093] Examples and Comparative Examples

[0094] Example 1

[0095] Example 1 uses p16 antibody to perform immunohistochemical staining on cervical tissue.

[0096] The DAB staining kit prepared in Example 1 was used as follows:

[0097] A1: Dewaxing:

[0098] a1. Bake the cut tissue chips at 65℃ for 30 minutes;

[0099] a2. After baking, directly immerse in xylene for dewaxing three times, 5 minutes each time;

[0100] A2: Hydration:

[0101] b1. After dewaxing, the sections were hydrated twice with 100% alcohol for 3 minutes each time, then hydrated with 95% alcohol for 3 minutes each time, and then hydrated with 85% alcohol for 3 minutes each time.

[0102] b2. Then, soak the hydrated slices in purified water for 2 minutes;

[0103] A3: Antigen thermal retrieval:

[0104] c1. Add EDTA repair solution with pH 9.0, heat it at 1800W on an induction cooker until the EDTA repair solution begins to boil, then add the hydrated slices, then reduce the induction cooker to 120W and let the EDTA repair solution boil intermittently for 20 minutes, then slowly cool to room temperature.

[0105] c2. Soak the repaired slices in distilled water for 2 minutes;

[0106] A4: Blocking endogenous peroxidase:

[0107] d1. Remove the liquid from the slide and use an immunohistochemistry pen to delineate the area of ​​tissue to be tested on the slide;

[0108] d2. Then wash the slides with PBST 4 times, 1 minute each time, taking care to prevent the slides from drying out;

[0109] d3. Add 100 μL of blocking agent evenly to each tissue section, ensuring the liquid covers the entire tissue, and incubate at room temperature for 10 min.

[0110] d4. Wash off the blocking agent and rinse 4 times with PBST solution, 1 min each time;

[0111] A5: Primary antibody incubation:

[0112] e1. Discard the PBST solution, and evenly drop 100uL of primary antibody working solution onto each slide, ensuring the liquid evenly covers the entire tissue. Incubate at room temperature for 30 minutes.

[0113] e2. After incubation, rinse 4 times with PBST solution, 1 minute each time;

[0114] A6: Incubation with reaction-enhancing solution:

[0115] f1. Discard the PBST solution, and evenly add 100 μL of reaction enhancement solution to each slide, ensuring the liquid evenly covers the entire tissue. Incubate at room temperature for 15 min.

[0116] f2. After incubation, rinse 4 times with PBST solution, 1 min each time;

[0117] A7: Secondary antibody incubation:

[0118] g1. Discard the PBST solution, and evenly add 100 μL of enzyme-labeled polymer IgG to each slide, ensuring the liquid evenly covers the entire tissue. Incubate at room temperature for 15 min.

[0119] g2. After incubation, rinse 4 times with PBST solution, 1 min each time;

[0120] A8: DAB color rendering:

[0121] h1. Discard the PBST solution, and evenly add 150 μL of freshly prepared DAB chromogenic solution using DAB buffer and DAB concentrate to each slide. The volume ratio of DAB buffer to DAB concentrate is 20:1. Develop the color at room temperature for 5 min.

[0122] h2. Observe the staining results. Generally, the color development time should not exceed 10 minutes. Then rinse with tap water for 3 minutes.

[0123] A9: Hematoxylin counterstaining:

[0124] j1. Remove the water from the tissue, then add 150 μL of hematoxylin staining solution for counterstaining and incubate for 1 min;

[0125] j2. Then immediately rinse it in tap water and soak it in tap water for 3 minutes to restore its blue color;

[0126] A10: Dehydration sealing sheet:

[0127] k1. After the slices turn blue, shake off the water, and then dehydrate them in 85% alcohol for 1 minute, 95% alcohol for 1 minute, and 100% alcohol twice for 1 minute each time.

[0128] k2. After removing the sections, bake them at 65℃ for 10 minutes. After drying, add neutral resin to seal the sections.

[0129] Example 2

[0130] The difference between Example 2 and Example 1 is that Example 2 uses the DAB staining kit prepared in Preparation Example 2.

[0131] Example 3

[0132] The difference between Example 3 and Example 1 is that Example 3 uses p120 antibody to perform immunohistochemical staining on breast cancer tissue.

[0133] Example 4

[0134] The difference between Example 4 and Example 1 is that Example 4 uses TTF-1 antibody to perform immunohistochemical staining on lung adenocarcinoma tissue.

[0135] Example 5

[0136] The difference between Example 5 and Example 1 is that Example 5 uses CK20 antibody to perform immunohistochemical staining on human colon tissue.

[0137] Example 6

[0138] The difference between Example 6 and Example 1 is that Example 6 uses an inhibin antibody to perform immunohistochemical staining on human placental tissue.

[0139] Comparative Example 1

[0140] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 uses the DAB staining kit prepared in Preparation Example 3.

[0141] Comparative Example 2

[0142] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 uses the DAB staining kit prepared in Preparation Example 4.

[0143] Comparative Example 3

[0144] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses commercially available DAB staining solution from Maixin Company, catalog number Kit-0014.

[0145] The kit consists of the following components:

[0146] Reagent 1: Endogenous peroxidase inhibitor

[0147] Reagent 2: Enzyme-labeled goat anti-mouse / rabbit IgG polymer

[0148] Reagent 3A: Enhanced DAB buffer (1x)

[0149] Reagent 3B: Enhanced DAB substrate (20x)

[0150] Reagent 3C: DAB chromogen (20x)

[0151] Comparative Example 3 used p120 antibody to perform immunohistochemical staining on breast cancer tissue.

[0152] The specific usage method is as follows:

[0153] 1. Dewaxing:

[0154] (1) Place the prepared slices in a 65℃ oven and bake for 2 hours.

[0155] (2) After baking, place the slices directly into xylene for dewaxing twice, each time for 5 minutes.

[0156] 2. Hydration:

[0157] (1) After dewaxing, the sections were hydrated twice with 100% alcohol for 3 minutes each time, then hydrated with 95% alcohol for 2 minutes, and then hydrated with 85% alcohol for 2 minutes.

[0158] (2) Then slice the ingredients and soak them in distilled water for 2 minutes.

[0159] 3. Antigen thermal repair

[0160] (1) Add EDTA repair solution with pH 9.0 to a stainless steel pot so that the slices can be completely immersed in the solution.

[0161] (2) Add EDTA repair solution with pH 9.0, heat it at 1800W on an induction cooker until the EDTA repair solution starts to boil, then put in the hydrated slices, then reduce the induction cooker to 120W and let the EDTA repair solution boil intermittently for 20 minutes, and then slowly cool it to room temperature.

[0162] (3) Soak the repaired slices in distilled water for 2 minutes.

[0163] 4. Blocking endogenous peroxidase

[0164] (1) Shake off the liquid on the slide and use an immunohistochemistry pen to circle the area of ​​the tissue to be tested on the slide.

[0165] (2) Then wash the slices with PBST 4 times, 1 min each time, taking care to prevent the slices from drying out.

[0166] (3) Add 2 drops (about 100ul) of endogenous peroxidase inhibitor to each tissue slice, spread it evenly to cover the entire tissue, and incubate at room temperature for 10min.

[0167] (4) After the time is up, wash off the blocking agent and rinse with PBST 4 times, 1 min each time.

[0168] 5. Primary antibody incubation

[0169] (1) Remove the PBS, add 2 drops (about 100ul) of the corresponding primary antibody working solution to each slide, spread it evenly to cover the entire tissue, and incubate at room temperature for 60min.

[0170] (2) After incubation, rinse with PBST 4 times, 1 min each time.

[0171] 6. Incubation with enzyme-labeled goat anti-mouse / rabbit IgG polymer

[0172] (1) Gently shake off the PBS, add 2 drops (about 100ul) of enzyme-labeled goat anti-mouse / rabbit IgG polymer to each slice, spread evenly to cover the entire tissue, and incubate at room temperature for 20min.

[0173] (2) After incubation, rinse with PBST 4 times, 1 min each time.

[0174] 7. DAB color rendering

[0175] (1) Gently shake off the PBS, add 150 μl of freshly prepared DAB chromogenic solution to each slice (DAB chromogen: enhanced DAB substrate: enhanced DAB buffer are prepared in a ratio of 1:1:20, and should be used immediately after preparation), and develop the color at room temperature for 3-5 min.

[0176] (2) Observe the staining results under a microscope. Generally, the staining time should not exceed 10 minutes. Rinse directly with tap water for 3 minutes.

[0177] 8. Hematoxylin counterstaining

[0178] (1) After rinsing with tap water, gently shake off the water on the tissue, then add 100-200 μl of hematoxylin staining solution for counterstaining and incubate for 30 seconds.

[0179] (2) After the time is up, immediately rinse it in tap water and soak it in tap water for 3 minutes to restore its blue color.

[0180] 9. Dehydration sealing sheet

[0181] (1) After the slices turn blue, shake off the water and then put them into 85% alcohol for 1 min, 95% alcohol for 1 min, and 100% alcohol twice for 1 min each time.

[0182] (2) After removing the slices, bake them at 65°C for 10 minutes. After drying, add neutral resin to seal the slices.

[0183] Comparative Example 4

[0184] Comparative Example 4 used commercially available DAB staining solution from Shanghai Gene Technology Co., Ltd., catalog number GK600505.

[0185] The kit consists of the following components:

[0186] Non-specific staining blocking agent (Solution A)

[0187] Enzyme-labeled goat anti-mouse / rabbit IgG polymer (Solution B)

[0188] DAB colorimetric solution (C1 solution)

[0189] Colorimetric buffer (C2 solution)

[0190] Comparative Example 4 used TTF-1 antibody to perform immunohistochemical staining on lung adenocarcinoma tissue.

[0191] The usage of Comparative Example 4 is as follows:

[0192] 1. Preparation of DAB working solution: Before the experiment, prepare the DAB working solution by adding 1 drop of DAB reagent (C1 solution) (approximately 40 μL) to each milliliter of colorimetric buffer (C2 solution). Mix well and then add the solution for color development. The DAB working solution should be prepared and used immediately. The prepared DAB working solution should be used within 2 hours. If precipitation occurs, mix well before use.

[0193] 2. Routine dewaxing and hydration: Immerse the paraffin sections in xylene three times, 5 minutes each time. Remove the sections and place them in 100% anhydrous ethanol twice, 3 minutes each time. Then, immerse them in 90%, 80%, and 70% ethanol for 3 minutes each. Rinse three times with PBS or TBS for 3 minutes each time.

[0194] 3. Antigen retrieval:

[0195] (1) Add EDTA repair solution with pH 9.0 to a stainless steel pot so that the slices can be completely immersed in the solution.

[0196] (2) Add EDTA repair solution with pH 9.0, heat it at 1800W on an induction cooker until the EDTA repair solution starts to boil, then put in the hydrated slices, then reduce the induction cooker to 120W and let the EDTA repair solution boil intermittently for 20 minutes, and then slowly cool it to room temperature.

[0197] (3) Soak the repaired slices in distilled water for 2 minutes.

[0198] 4. After antigen retrieval is complete and the slides have cooled naturally, rinse them three times with PBS or TBS for 3 minutes each time.

[0199] 5. Remove the slide, shake off and wipe away the liquid around the tissue, and add a drop of nonspecific staining blocking agent (solution A) (usually 3% hydrogen peroxide) to the tissue and incubate in the dark for 10-15 minutes. Rinse three times with PBS or TBS, 3 minutes each time. Remove the slide, shake off and wipe away the liquid around the tissue (do not let the tissue dry).

[0200] 6. Add 80 μL of primary antibody working solution (rabbit or mouse antibody) to the tissue and incubate (follow the instructions for each primary antibody). After primary antibody incubation, rinse the sections with TBS or PBS. Rinse three times with PBS or TBS for 3 minutes each time, remove the sections, shake off and wipe away the liquid around the tissue (do not dry the tissue).

[0201] 7. Add 80 μL of secondary antibody B solution (enzyme-labeled goat anti-mouse / rabbit IgG polymer) to the tissue and incubate at room temperature for 25 min. After incubation, place the slide in TBS or PBS and rinse 3 times for 3 min each time. Remove the slide, shake off and wipe the liquid around the tissue (do not dry the tissue).

[0202] 8. Add 80 μL of the prepared fresh DAB working solution as a colorimetric reagent and incubate at room temperature for 8 minutes, or control the color development under a light microscope. After complete color development, rinse with distilled water to stop the color development process.

[0203] 9. Rinse with tap water and then redye with hematoxylin.

[0204] 10. Dehydrate using 70%, 80%, 90%, and 100% alcohol solutions for 3 minutes each time. Remove the sections and immerse them in xylene three times for 5 minutes each time.

[0205] 11. The sections are dehydrated, cleared, and then mounted using standard procedures.

[0206] Comparative Example 5

[0207] Comparative Example 5 uses the old formulation of DAB buffer.

[0208] The old formula for DAB buffer was as follows: Add 1 mL of 0.1% hydrogen peroxide, 3.4 g of 50 mM imidazole, 1 g of 0.1% nonylphenol polyoxyethylene ether, and 100 μL of 0.01% chloroalkyl dimethylbenzylamine to 1000 mL of purified water and mix thoroughly.

[0209] The usage method of Comparative Example 5 is the same as that of Example 1.

[0210] Comparative Example 5 used CK20 antibody to perform immunohistochemical staining on human colon tissue.

[0211] Comparative Example 6

[0212] The difference between Comparative Example 6 and Comparative Example 5 is that Comparative Example 6 uses an inhibin antibody to perform immunohistochemical staining on human placental tissue.

[0213] Comparative Example 7

[0214] The difference between Comparative Example 7 and Comparative Example 3 is that Comparative Example 7 uses p16 antibody to perform immunohistochemical staining on cervical tissue.

[0215] Example 7

[0216] After preparing the DAB chromogenic solution by diluting the DAB concentrate and DAB buffer in the DAB staining kit of the present invention at a ratio of 1:20, and storing it at 2-8℃ for 0 days, 3 days and 7 days respectively, the tonsil tissue was then subjected to immunohistochemical staining using CD8 antibody.

[0217] Performance testing

[0218] 1. Observe the staining results of the above examples and comparative examples under a microscope at 100x field of view. The results are shown in the figure.

[0219] From Example 1 and Comparative Example 7 ( Figure 1 Immunohistochemical staining results of cervical tissue with p16 antibody showed that, compared with the commercially available DAB staining solution Kit-0014 from Maixin Company, the DAB staining solution kit of this invention has stronger staining intensity and higher sensitivity.

[0220] From Example 3 and Comparative Example 3 ( Figure 2 Immunohistochemical staining results of p120 antibody on breast cancer tissue show that the staining intensity of the DAB staining solution kit of this invention is significantly stronger and the staining sensitivity is higher than that of the commercially available DAB staining solution Kit-0014 from Maixin Company, and there is no background staining.

[0221] From Example 4 and Comparative Example 4 ( Figure 3 Immunohistochemical staining results of TTF-1 antibody on lung adenocarcinoma tissue showed that the staining intensity of the DAB staining solution kit of this invention was significantly stronger than that of the commercially available DAB staining solution from Shanghai Gene Technology Co., Ltd. (product number GK600505), with higher staining sensitivity and no background staining.

[0222] From Example 5 and Comparative Example 5 ( Figure 4 Immunohistochemical staining results of human colon tissue with CK20 antibody showed that the staining effect of the DAB staining solution kit of the present invention is significantly better than that of the old DAB staining solution, and the color development speed is faster and the required color development time is shorter.

[0223] From Example 6 and Comparative Example 6 ( Figure 5 Immunohistochemical staining results of human placental tissue with the inhibitor antibody in this invention show that the staining effect of the DAB staining solution kit of this invention is significantly better than that of the old DAB staining solution, and the color development speed is faster and the required color development time is shorter.

[0224] from Figure 6-8 It can be seen that after the DAB colorimetric solution prepared in this application was placed at 2-8℃ for 0 days, 3 days and 7 days, the colorimetric effect of the DAB colorimetric solution within 7 days was not significantly different, the colorimetric quality was very good, and there was no significant decrease in colorimetric effect after a long time, indicating good stability.

[0225] from Figure 9 It can be seen that after 10 seconds, the color development effect of the DAB color development solution of this application is better than that of the old DAB color development solution, and the color development speed is faster and the required color development time is shorter. Furthermore, the old DAB color development solution no longer has a staining effect (negative) after 7 days.

[0226] In summary, the DAB staining kit of this application exhibits faster color development speed, shorter color development time, and good stability of the prepared DAB chromogenic solution. This is because the metal ions in the DAB buffer enhance the electron transfer rate between DAB and H₂O₂, resulting in a shorter reaction time, higher color development efficiency, and ultimately improved sensitivity of DAB staining. The enzyme-labeled polymer IgG in the DAB staining kit disclosed in this application has high sensitivity and specificity. When used with the DAB concentrate and buffer in the kit, it achieves extremely high staining quality and is relatively inexpensive, making it a promising candidate for application in the in vitro diagnostics industry. Furthermore, the DAB buffer and DAB concentrate, when mixed at a 1:20 ratio, remain unaffected after 7 days at 2-8°C, maintaining good staining performance. This reduces the frequency of preparation by laboratory personnel and allows for more flexible experimental scheduling. Therefore, the DAB staining kit of this application improves the detection sensitivity of immunohistochemical staining while also enhancing the storage stability of the DAB chromogenic solution and reducing costs.

[0227] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A DAB buffer solution for DAB color development, characterized in that: The DAB buffer solution is composed of hydrogen peroxide, imidazole, nonylphenol polyoxyethylene ether, chloroalkyl dimethylbenzylamine, magnesium sulfate and trisodium citrate. The volume concentration of the hydrogen peroxide is 0.05-0.3%. The molar concentration of the imidazole is 30-80 mM. The nonylphenol polyoxyethylene ether has a mass percentage concentration of 0.04-0.18%. The volume concentration of the chloroalkyl dimethylbenzylamine is 0.006-0.015%. The molar concentration of the magnesium sulfate is 0.5-2.2 mM. The molar concentration of the trisodium citrate is 0.6-1.8 mM.

2. A DAB staining reagent kit, comprising an inhibitor, a reaction enhancement solution, enzyme-labeled polymer IgG, DAB concentrate, DAB buffer, and hematoxylin, characterized in that: The DAB buffer is the DAB buffer for DAB color development as described in claim 1.

3. The DAB staining reagent kit according to claim 2, characterized in that: The blocking agent is a hydrogen peroxide solution with a concentration of 1-6%.

4. The DAB staining reagent kit according to claim 2, characterized in that: The main component of the reaction enhancement solution is rabbit anti-mouse immunoglobulin.

5. The DAB staining reagent kit according to claim 4, characterized in that: The concentration of the reaction enhancement solution is 130-550 ug / mL.

6. The DAB staining reagent kit according to claim 2, characterized in that: The DAB concentrate is prepared from 1,2-propanediol, water and DAB.

7. The DAB staining reagent kit according to claim 2, characterized in that: The enzyme-labeled polymer IgG was obtained by the following preparation method: S1. Mix 3-15 mg / mL of goat anti-rabbit antibody and 2-10 mg / mL of goat anti-mouse antibody evenly, then add 12-26 mg / mL of ethylene glycol chitosan polymer and mix evenly. Then add 20-50 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3-8 mg / mL of N-hydroxysulfosuccinimide. Mix well and adjust the pH to 2.2-5.4 with 0.03-0.2 M HCl. React at room temperature in the dark for 2-8 hours to obtain solution A for later use. S2. Add 6-16 mg / mL of peroxidase solution to 18-30 mg / mL of polyethylene glycol 2000 solution, then add 2-8 mg / mL of polyethylene glycol-modified SMCC crosslinking agent, react in the dark for 2-8 hours to obtain solution B for later use. S3. Mix the above solutions A and B, add 1-4 mg / mL of polyethylene glycol-modified SMCC crosslinking agent, adjust the pH to 6.5-7.5, react in the dark for 2-6 hours, and obtain the product after adsorption and elution.

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