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For easy results with DAB staining, use a simple DAB substrate kit, or complete DAB staining kit, such as streptavidin-biotin HRP/DAB kits, and polymer method HRP/DAB kits. Alternatively, you can assemble the reagents for DAB staining yourself, using biotin-conjugated secondary antibodies, HRP-polymer-conjugated secondary antibodies, and streptavidin-HRP conjugates.
DAB (3,3′-Diaminobenzidine) is a derivative of benzene. It is most often used in immunohistochemical (IHC) staining as a chromogen. It is also used in in situ hybridization (ISH) and sometimes in dot blots and in western blotting.
In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP). The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be visualized using light microscopy.
The DAB precipitate is insoluble in water, alcohol, and other organic solvents most commonly used in the lab (such as xylene and isopropanol). This allows for great flexibility in the subsequent treatment of the tissue section, such as in the choice of counterstain and mounting medium. DAB staining is also heat-resistant, so it can be used in double labeling IHC/ISH experiments, and is extremely stable – in fact stained slides are often stable for many years.
When used together with a nickel or cobalt solution as a DAB enhancer, DAB staining becomes a more intense, black color.
In IHC, there are three main methods of linking HRP to the primary antibody used for staining. These have the following advantages and disadvantages:
- Directly HRP-conjugated primary antibodies
Less amplification, and thus less sensitive than following methods. Very simple protocol. Requirement for HRP conjugation of primary antibody.
- Biotinylated secondary antibodies, combined with streptavidin-HRP in the ABC (Avidin Biotin Complex) method
Requirement for blocking of endogenous biotin to avoid background staining. Most heavily used method in research labs.
- HRP-polymer secondary antibodies
Shorter protocol than the ABC method. Highly sensitive with low background. Abcam uses this method for the validation of antibodies in IHC.
Jovicic N et al used ab64259 HRP/DAB streptavidin-biotin ABC method kit to stain CD68 in paraffin-embedded mouse liver sections. Brown DAB staining shows the presence of CD68.
Compared to fluorescent methods used for IHC, chromogenic detection, such as DAB staining, is typically more sensitive due to the enzymatic amplification used with DAB staining and similar methods. However, it is not possible to resolve sub-cellular structures using DAB staining to the same degree as with fluorescent microscopy.
Although DAB is overwhelmingly the most popular chromogen, there alternatives. See our guide to chromogens and enhancers for full details on different chromogens and their advantages and disadvantages.
A disadvantage of DAB is that it reacts slowly with hydrogen peroxide, even without the presence of a peroxidase enzyme. This means that once the DAB is combined with hydrogen peroxide, it should be used relatively quickly to avoid generating brown precipitate in the solution, which will contribute to background staining. Typically, kits for DAB staining are supplied with concentrated DAB substrate, and a separate buffer containing hydrogen peroxide, to be combined shortly before use.
Be aware that DAB is toxic – it is a suspected teratogen and carcinogen, so it should be handled according to good laboratory practice.
For complete protocols for immunostaining using DAB, please see our collection of validated IHC protocols based on our own experience with antibody validation.
A lot of DAB staining uses pre-prepared DAB solutions, such as in DAB substrate kit ab64238. It is also possible to prepare DAB using DAB tablets. Tablets are generally used rather than DAB powder to minimize health and safety risks in preparing the DAB solution.
Whether using tablets or pre-prepared solutions, read the manufacturer’s instructions for preparation as these can vary, for instance if hydrogen peroxide is already included or must be added.
Once the DAB solution is prepared with hydrogen peroxide included, and after sections have been stained with HRP, the sections simply need to be incubated with the DAB solution for a few minutes, followed by rinsing in either a buffer, such as PBS, or in water. DAB staining is usually followed by counterstaining, dehydration in an ethanol series and mounting.
To avoid excessive staining with DAB, it is best to monitor the staining process under a microscope and remove the DAB when a light brown staining is seen.
Jovicic N et al. PLoS ONE 10: e0134089 (2015)