Tablets are generally used rather than DAB powder to minimize health and safety risks in preparing the DAB solution. It is also possible to prepare DAB using DAB tablets. DAB staining protocolsįor complete protocols for immunostaining using DAB, please see our collection of validated IHC protocols based on our own experience with antibody validation.Ī lot of DAB staining uses pre-prepared DAB solutions, such as in DAB substrate kit ab64238. Typically, kits for DAB staining are supplied with concentrated DAB substrate, and a separate buffer containing hydrogen peroxide, to be combined shortly before use.īe aware that DAB is toxic – it is a suspected teratogen and carcinogen, so it should be handled according to good laboratory practice. 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. Pitfalls to avoid with DAB stainingĪ disadvantage of DAB is that it reacts slowly with hydrogen peroxide, even without the presence of a peroxidase enzyme. See our guide to chromogens and enhancers for full details on different chromogens and their advantages and disadvantages. However, it is not possible to resolve sub-cellular structures using DAB staining to the same degree as with fluorescent microscopy.Īlthough DAB is overwhelmingly the most popular chromogen, there alternatives. Alternatives to DAB stainingĬompared 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. Brown DAB staining shows the presence of CD68. Jovicic N et al used ab64259 HRP/DAB streptavidin-biotin ABC method kit to stain CD68 in paraffin-embedded mouse liver sections. Abcam uses this method for the validation of antibodies in IHC. Most heavily used method in research labs. Requirement for blocking of endogenous biotin to avoid background staining. Biotinylated secondary antibodies, combined with streptavidin-HRP in the ABC (Avidin Biotin Complex) method Requirement for HRP conjugation of primary antibody. Less amplification, and thus less sensitive than following methods. Directly HRP-conjugated primary antibodies These have the following advantages and disadvantages: In IHC, there are three main methods of linking HRP to the primary antibody used for staining. Linking HRP and primary antibodies for DAB staining When used together with a nickel or cobalt solution as a DAB enhancer, DAB staining becomes a more intense, black color. 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. This allows for great flexibility in the subsequent treatment of the tissue section, such as in the choice of counterstain and mounting medium. The DAB precipitate is insoluble in water, alcohol, and other organic solvents most commonly used in the lab (such as xylene and isopropanol). The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be visualized using light microscopy. In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP). It is also used in in situ hybridization (ISH) and sometimes in dot blots and in western blotting. It is most often used in immunohistochemical (IHC) staining as a chromogen. Principles and application of DAB stainingĭAB (3,3′-Diaminobenzidine) is a derivative of benzene. Alternatively, you can assemble the reagents for DAB staining yourself, using biotin-conjugated secondary antibodies, HRP-polymer-conjugated secondary antibodies, and streptavidin-HRP conjugates. Linking HRP and primary antibodies for DAB stainingįor 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.Principles and application of DAB staining.
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