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Classification of HRP substrates

Time：

2023-05-16

The substrate range is wide and includes diaminobenzidine (DAB), chloronaphthol and aminomethylcarbazole.

The wide range of substrates includes diaminobenzidine (DAB), chloronaphthol and aminoethylcarbazole.

　　Diaminobenzidine/metal salts

　　DAB is the most sensitive and commonly used substrate for horseradish peroxidase. It produces an intense brown product that is insoluble in water and alcohol. In most cases, the addition of different metal ions to DAB is recommended. Horseradish peroxidase can be more sensitive when metal salts such as cobalt and nickel are added to the substrate solution. Such reaction products are dark blue-gray to black in color and stable in water and alcohols.DAB/metal salt staining is applied to a wide range of tissues.

　　1. Required solutions and special equipment

　　DAB, 0.05 mol/L Tris buffer (pH 7.6), 0.3% (W/V) nickel chloride/water storage solution, 30% hydrogen peroxide, DPX, optical microscope (usually with camera for recording results).

　　2. Procedure

　　(1) Dissolve 6 mg of DAB in 9 mL of 0.05 mol/L Tris buffer (pH 7.6).

　　(2) Add l mL of 0.3% (W/V) NiCl2/water storage solution (equivalent amount of cobalt chloride can be used instead).

　　(3) Add 0.1 mL of 3% hydrogen peroxide. Hydrogen peroxide is generally provided in a 30% solution and stored at 4 °C. This condition can last up to 1 month.

　　(4) If precipitation occurs, filter through filter paper.

　　(5) Add this solution to the specimen and terminate the reaction by incubating for 1 to 20 min with water.

　　(6) Optimize: if needed, replicate staining.

　　(7) Seal the slides with DPX.

　　Clonaphthol

　　Chloronaphthol can form a blue-black product. It is less sensitive than DAB and the product is soluble in alcohol. It is suitable when DAB reaction forms a higher background or is required to change the color of the product.

　　1. Solution and special equipment required

　　0.03% chloronaphthol, dissolved in anhydrous ethanol (stored at -20°C), 0.05 mol/L Tris (pH 7.6), hydrogen peroxide, Gelvatol or Mowiol, optical microscope (usually with a camera to facilitate recording of results).

　　2. Procedure

　　(1) Preparation of chloronaphthol storage solution: dissolve 0.3 g of chloronaphthol in 10 mL of anhydrous ethanol and store at -20°C.

　　(2) Add 100 μL of chloronaphthol storage solution to 10 mL of 0.05 mol/LTris (pH 7.6).

　　(3) Add 0.1 mL of 3% hydrogen peroxide to water. Hydrogen peroxide is generally available as a 30% solution and can be stored at 4°C for 1 month.

　　(4) The white precipitate can be removed by filtering through filter paper.

　　(5) Add this solution to the specimen and incubate at room temperature for 10-40 min, and terminate the reaction by washing with water.

　　(6) Optimization: If needed, perform a re-staining, but only water-soluble dyes should be used.

　　(7) Seal the slices with Gelvatol or Mowiol.

　　Aminoethylcarbazole

　　Aminoethylcarbazole (AEC) produces a red product that is less sensitive than DAB, but can be used in experiments where the DAB reaction produces a higher background or where a change in product color is required. This product is soluble in alcohols but insoluble in water.

　　1. Solutions and special equipment required

　　0.4% AEC dissolved in N,N-dimethylformamide (DMF), 0.1 mol/L sodium acetate buffer (pH 5.2), hydrogen peroxide, Gelvatol or Mowiol, optical microscope (usually with a camera to facilitate recording of results).

　　2. Procedure

　　(1) Dissolve 4 mg of AEC in l mL of DMF. AEC is stable in DMF, so 100 mL of DMF can be used to dissolve 0.4 g of AEC as a storage solution.

　　(2) Add l mL of AEC solution to 15 mL of 0.1 mol/L sodium acetate buffer (pH 5.2) and stir.

　　(3) Add 0.15 mL of 3% hydrogen peroxide to water. Hydrogen peroxide is generally provided in 30% solution and can be stored at 4°C for 1 month.

　　(4) Filter through filter paper.

　　(5) Add this solution to the specimen and incubate at room temperature for 10-40 min, terminate the reaction by washing with water.

　　(6) Optimization: If re-staining is required, use only water-soluble stain