DAB-4HCl (3,3′-diaminobenzidine tetrahydrochloride) is a chromogenic substrate used for horseradish peroxidase (HRP) detection in immunoperoxidase, immunohistochemical, and histochemical workflows. Oxidation of DAB in the presence of peroxidase and hydrogen peroxide produces a localized, insoluble brown reaction product that provides strong contrast at sites of enzyme activity.
Synonyms: DAB-4HCl; 3,3′-Diaminobenzidine tetrahydrochloride; 3,3′,4,4′-Tetraaminobiphenyl tetrahydrochloride.
Cat. 04008 is supplied as 10 mg of lyophilized DAB-4HCl per vial. The preweighed vial format reduces direct handling of the solid reagent during solution preparation. Polysciences Technical Data Sheet 164 describes preparation of a 0.5 mg/mL DAB solution by adding the appropriate buffer directly to the vial.
The oxidized DAB reaction product is also osmiophilic. Subsequent osmication increases electron density, allowing localized reaction product to be examined in electron microscopy and correlated light-electron microscopy workflows where an appropriate validated protocol is used.
Key Properties
- Chromogenic substrate for horseradish peroxidase detection
- Immunochemical Grade
- CAS Number: 7411-49-6
- Linear formula: C12H14N4 · 4HCl
- Molecular weight: approximately 360.1 g/mol
- Physical form: lyophilized solid
- Preweighed at 10 mg per vial
- Package size: 5 × 10 mg vials
- Catalog number: 04008
Applications
HRP Immunoperoxidase Staining
DAB-4HCl is used as a chromogenic substrate for HRP-based immunoperoxidase methods. In the presence of hydrogen peroxide, HRP catalyzes oxidation of DAB to form an insoluble brown reaction product at the site of enzyme localization.
Immunohistochemistry & Immunocytochemistry
The localized DAB reaction product provides visible contrast for HRP-based detection of antigens or antibodies in tissue and cell preparations. Staining intensity and localization depend on the complete immunochemical protocol, including antibody specificity, HRP activity, substrate concentration, incubation time, and tissue preparation.
Histochemical Peroxidase Localization
DAB-4HCl has also been used to demonstrate endogenous peroxidase and peroxidatic enzyme activity in histochemical studies. Appropriate controls are required to distinguish endogenous activity from exogenous HRP labeling.
Electron Microscopy
DAB oxidation products are osmiophilic and can be further treated with osmium tetroxide to increase electron opacity. This property supports ultrastructural localization of peroxidase-associated reaction products in appropriately designed electron microscopy workflows.
Compatibility & Bonding Considerations
- DAB-4HCl functions as an electron donor in HRP-catalyzed reactions with hydrogen peroxide
- Oxidation produces insoluble polymeric reaction products that remain localized at sites of enzymatic activity
- Reaction intensity depends on HRP activity, hydrogen peroxide concentration, DAB concentration, pH, temperature, and incubation time
- Endogenous peroxidase activity can contribute background signal and should be addressed with appropriate controls when relevant
- Osmication can increase electron density of the DAB reaction product for ultrastructural imaging
- Optimization should be performed for the specific antibody, specimen, fixation method, and detection system
DAB staining intensity should not be interpreted solely as a direct measure of antigen abundance unless the complete assay has been appropriately validated for quantitative comparison.
Application Tips
- Technical Data Sheet 164 describes preparation of DAB-4HCl at 0.5 mg/mL in an appropriate buffer
- Add buffer directly to the preweighed vial to minimize handling of the dry reagent
- Prepare the DAB working solution according to the selected validated immunoperoxidase or histochemical protocol
- Use consistent DAB concentration, hydrogen peroxide concentration, incubation time, and temperature when comparing samples
- Include appropriate positive, negative, and no-primary-antibody controls for immunochemical staining
- Use freshly prepared working solution when required by the selected protocol
Handling & Storage
- Protect from moisture
- Store at −20°C according to the current product-page storage instruction
- Handle in a chemical fume hood or other well-ventilated laboratory area
- Avoid breathing dust, mist, or aerosol
- Avoid contact with skin and eyes
- Wear protective gloves, safety glasses, and suitable protective clothing
Why Choose Polysciences?
Trusted by 6,000+ Researchers Worldwide
Cited in Thousands of Peer-Reviewed Studies
U.S. Manufacturing & Global Distribution
Reliable Delivery to 40+ Countries
FAQ
Click a question to expand.
1. What is DAB-4HCl used for?
DAB-4HCl is used as a chromogenic substrate for horseradish peroxidase in immunoperoxidase, immunohistochemical, immunocytochemical, and histochemical staining methods. Oxidation produces a localized brown reaction product.
2. What is the full chemical name of DAB-4HCl?
DAB-4HCl is 3,3′-diaminobenzidine tetrahydrochloride, CAS Number 7411-49-6. It is also described as 3,3′,4,4′-tetraaminobiphenyl tetrahydrochloride.
3. How is DAB used with horseradish peroxidase?
In an HRP detection system, DAB serves as an electron donor and hydrogen peroxide serves as the oxidizing substrate. HRP catalyzes formation of an insoluble brown DAB oxidation product at the site of enzyme activity.
4. Can DAB reaction product be examined by electron microscopy?
Yes. Oxidized DAB products are osmiophilic. Subsequent osmication can increase electron density, allowing localized reaction product to be visualized in appropriately designed electron microscopy workflows.
5. What is included with Cat. 04008?
Cat. 04008 is supplied as five vials, each containing 10 mg of lyophilized DAB-4HCl. The preweighed vial format reduces direct handling of the dry reagent during solution preparation.
Safety & Documentation
DAB-4HCl is toxic if swallowed or in contact with skin, harmful if inhaled, causes eye irritation, and is suspected of causing cancer. Handle only after reviewing the Safety Data Sheet, use appropriate laboratory ventilation, and wear protective gloves, eye protection, and suitable protective clothing.
