What is 3,3' Diaminobenzidine?
3,3' Diaminobenzidine (DAB) is a chromogen commonly used in histology for the visualization of cellular and tissue components. It is a substrate for peroxidase enzymes, particularly horseradish peroxidase (HRP), and upon oxidation, it produces a brown precipitate that can be visualized under a microscope.
How is DAB used in Immunohistochemistry?
In
immunohistochemistry (IHC), DAB is often used to detect the presence of specific antigens in tissue sections. The process involves the application of an
antibody conjugated to HRP to the tissue sample. When DAB is added, the HRP catalyzes its oxidation, resulting in a brown stain at the site of the antigen. This allows researchers to observe and analyze the distribution and localization of proteins within the tissue.
It produces a stable and permanent brown color that is easily visible under standard light microscopy.
The staining is highly specific, allowing for precise localization of target antigens.
It has low background staining, which improves the contrast and clarity of the results.
DAB can be used in conjunction with other chromogens for multiplexing, enabling the simultaneous visualization of multiple targets.
DAB is a potential carcinogen, so appropriate safety measures, such as wearing gloves and working in a well-ventilated area, are necessary.
The brown precipitate can sometimes obscure other tissue details, making it challenging to interpret results in complex samples.
DAB staining can fade over time, although this is generally minimal compared to other chromogens.
It is important to optimize the staining protocol to avoid non-specific binding and ensure reproducibility.
Prepare the tissue sections and perform antigen retrieval if necessary.
Block endogenous peroxidase activity to prevent non-specific staining.
Apply the primary antibody specific to the target antigen.
Add a secondary antibody conjugated to HRP.
Incubate the tissue with the DAB substrate solution.
Monitor the development of the brown color under a microscope.
Stop the reaction by rinsing with water and counterstain if desired.
Dehydrate, clear, and mount the tissue sections for microscopic examination.
Cancer research: Identifying and localizing tumor markers in tissue samples.
Neuroscience: Visualizing the distribution of neurotransmitters and receptors in brain tissue.
Pathology: Diagnosing diseases by examining the presence of specific proteins or pathogens.
Developmental biology: Studying the expression patterns of proteins during embryonic development.
