DAB Peroxidase Substrate Kit (Brown)
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MCE DAB Peroxidase Substrate Kit (Brown) is a chromogenic reagent kit based on the HRP-catalyzed oxidation of DAB. It is suitable for staining and chromogenic detection in immunohistochemistry, immunocytochemistry, in situ hybridization, Western blotting, and other applications using cell or tissue samples. The resulting insoluble brown precipitate provides clear localization and stable visualization of target signals, facilitating direct observation and analysis. Following DAB development, counterstaining can be performed according to experimental requirements. For example, ethanol-soluble dyes can be used for subsequent counterstaining to facilitate the visualization and analysis of cellular or tissue morphology.
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Storage :
DAB Chromogen: -20°C, 1 year. Keep away from light and avoid repeat freeze-thaw cycles. DAB Buffer, DAB Oxidizer: 4°C, 1 year. Keep away from light.
- Manual (3577KB)
- COA
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Description & Advantages
3,3′-Diaminobenzidine (DAB) is a commonly used chromogenic substrate for horseradish peroxidase (HRP) and is widely used for colorimetric detection in immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization, Western blotting, and other applications. In the presence of HRP, DAB undergoes an oxidation reaction with hydrogen peroxide (H2O2), generating an insoluble brown precipitate that enables visualization of HRP-labeled signals.
MCE DAB Peroxidase Substrate Kit (Brown) is a chromogenic reagent kit based on the HRP-catalyzed oxidation of DAB. It is suitable for staining and chromogenic detection in immunohistochemistry, immunocytochemistry, in situ hybridization, Western blotting, and other applications using cell or tissue samples. The resulting insoluble brown precipitate provides clear localization and stable visualization of target signals, facilitating direct observation and analysis. Following DAB development, counterstaining can be performed according to experimental requirements. For example, ethanol-soluble dyes can be used for subsequent counterstaining to facilitate the visualization and analysis of cellular or tissue morphology.
Protocol
1. Sample Pretreatment and Washing: After conventional tissue sections, cell samples, or membranes have been incubated with horseradish peroxidase (HRP)-labeled antibodies or other probes, wash the samples 3-5 times with an appropriate washing buffer. For tissue or cell samples used to detect endogenous peroxidase activity, wash 3-5 times with washing buffer after appropriate fixation, 3-5 min each time.
2. Preparation of DAB Working Solution: Prepare the DAB working solution according to the table below.
| Component | Volume |
|---|---|
| DAB Chromogen Solution | 20 mL |
| DAB Buffer | 20 mL |
| DAB Oxidant | 20 mL |
| DAB Working Solution (Total Volume) | 200 mL |
Note: It is recommended to prepare the DAB working solution immediately before use. Prolonged storage may affect color development.
3. DAB Color Development: After washing, completely remove the washing buffer and add a sufficient volume of DAB working solution to fully cover the sample. Incubate at room temperature in the dark for 1-30 min, or extend the incubation time as required until the desired staining intensity is achieved.
Note: a. If no obvious background staining is observed, the color development time may be appropriately extended to achieve the desired staining intensity.
b. For Western Blot experiments, add a sufficient volume of DAB working solution and ensure that the membrane can move freely in the solution to achieve uniform color development.
4. Termination of Color Development: Remove the DAB working solution and wash thoroughly with ddH2O to remove residual chromogen solution and terminate the color-development reaction.
5. Subsequent Processing and Storage: For cell samples or tissue sections observed by microscopy, counterstaining with Neutral Red may be performed after color development according to experimental requirements to facilitate visualization of cellular or tissue morphology. For Western Blot experiments, rinse the membrane with ddH2O after color development to remove residual chromogen solution, then air-dry naturally. Store the dried membrane at room temperature protected from light.
Storage
DAB Chromogen: -20°C, 1 year. Keep away from light and avoid repeat freeze-thaw cycles.
DAB Buffer, DAB Oxidizer: 4°C, 1 year. Keep away from light.
Attention
1. Sodium azide (NaN3) inhibits HRP activity. Therefore, sodium azide (NaN3) should be avoided in the reaction system to prevent interference with the chromogenic reaction.
2. The DAB Oxidizer is volatile and should be tightly sealed during storage to prevent reagent performance from being compromised by evaporation. Use promptly after opening.
3. DAB is harmful to human health. Appropriate personal protective equipment should be worn during handling. Avoid direct contact with the skin or eyes and avoid inhalation or ingestion.;
4. The development time may be adjusted according to the actual experimental conditions to avoid insufficient or excessive staining.
5. This product is for R&D use only, not for drug, household, or other uses.
6. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Cat. No. | Product List | Components | HY-K1106-100 mL | Storage |
|---|---|---|---|---|
| HY-K1106-A | DAB Reagent A | DAB Chromogen | 50 mL | -20°C, 1 year. Keep away from light and avoid repeat freeze-thaw cycles. |
| HY-K1106-B | DAB Reagent B | DAB Buffer | 50 mL | 4°C, 1 year. Keep away from light. |
| DAB Oxidizer | 100 μL |
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