Vivian Marlowe
Chromogens are used in immunohistochemistry (IHC) and in situ hybridization (ISH) to detect target biomarkers in tissue samples. Traditional chromogens such as 3,3'- diaminobenzidine (DAB) produce a dark brown precipitate that is insoluble in alcohol. However, Fast Red-based chromogens are prone to fading and/or blushing when exposed to alcohol. The choice of chromogen depends on the specific application and the presence of tissue pigmentation.
| Characteristics | Values |
|---|---|
| Traditional chromogens | DAB, Fast Red-based chromogens |
| Innovative chromogens | Purple, Green, Yellow, Blue, Teal |
| High-contrast chromogens | Silver Stain |
| Insoluble in alcohol | 3,3’ diaminobenzidine |
| Nitro-blue tetrazolium/5-bromo-4-chloro-3’-indolyphosphate (NBT/BCIP) |
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What You'll Learn
DAB chromogen
DAB, or 3,3’-Diaminobenzidine, is a widely used chromogen for immunohistochemical staining and immunoblotting. When used in conjunction with a peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol and xylene. This precipitate provides a nice contrast to Hematoxylin counterstains, allowing for easy visualisation of targets.
The HIGHDEF® DAB Chromogen/Substrate Set is a popular product that makes use of DAB. This product can be used in conjunction with peroxidase-based immunostaining systems. Peroxidase from the antibody detection system reacts with hydrogen peroxide substrate to degrade it, which then reacts with DAB, precipitating it at positive sites and yielding a dark brown colour.
DAB is well accepted among pathologists because of its increased sensitivity and decreased background when compared to amino ethyl carbazole (AEC). Specimens stained with DAB can be dehydrated, cleared, and mounted for permanent record-keeping. The HIGHDEF® DAB Chromogen/Substrate Set is more sensitive and stable than traditional working DAB solutions.
In terms of its use, DAB is traditionally the main chromogen of choice for general applications, with Red following close behind for tissues with pigmentation. However, if the tissue being analysed has natural pigmentation, such as endogenous melanin protein, DAB can be hard to differentiate from the melanin in the tissue.
In summary, DAB is a commonly used chromogen that produces an alcohol-insoluble brown precipitate when reacted with a peroxidase enzyme. It is favoured for its sensitivity and stability, and its ability to provide good contrast for visualisation.
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Fast Red chromogen
Fast Red is a widely used chromogen for immunohistochemical staining with alkaline phosphatase (AP) detection systems. When reacted with alkaline phosphatase, Fast Red produces an insoluble red precipitate at the reaction site on tissues and cells. AP hydrolyzes the naphthol phosphate esters to phenolics and phosphates. The phenols then couple with colorless diazonium salts to produce an insoluble coloured azo dye.
The Fast Red Chromogen Kit consists of two solutions for the staining of formalin-fixed, paraffin-embedded (FFPE) tissues, as part of an immunohistochemistry (IHC) procedure using an alkaline phosphatase (AP) detection system. Immunohistochemistry (IHC) permits the visual identification of specific protein antigens in tissues for diagnostic purposes. Following application of the primary antibody, the presence of a target antigen is visualized by the sequential application of an enzyme-antibody conjugate that binds the primary antibody, and a chromogen reagent, to produce a coloured reaction product that is visible by light microscopy.
The Vulcan Fast Red chromogen is a stable two-component system, consisting of Vulcan Fast Red Chromogen and Vulcan Buffer. When using an alkaline phosphatase system, tris buffer (pH 7.6) should be used as a rinsing buffer. PBS should never be used as phosphates act as a competitive inhibitor to alkaline phosphatase enzymes.
Fast Red-based chromogens have a high-contrast colour but are prone to fading and/or blushing when exposed to alcohol or xylene. Traditional chromogens such as DAB and Fast Red-based chromogens have very broad absorption spectra. Chromogens that have narrower absorption ranges can take on translucent qualities.
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Silver staining chromogen
Silver staining is used to stain gels. The silver staining of proteins in Agarose gels was developed in 1973 by Kerenyi and Gallyas. It was later adapted for polyacrylamide gels used in SDS-PAGE, and also for staining DNA or RNA. The glycosylations of glycoproteins and polysaccharides can be oxidised by a 1-hour pre-treatment with 0.1% periodic acid at 4 °C, which improves the binding of silver ions and the staining result.
Firstly, the proteins are denatured in the gel by a fixative solution of 10% acetic acid and 30% ethanol and precipitated, while the detergent (mostly SDS) is extracted. The diffusion of the proteins is thus significantly reduced. After repeated washing with water, the gel is incubated in a silver nitrate solution. Silver nitrate forms insoluble silver phosphate with phosphate ions; this method is known as the Von Kossa Stain. When subjected to a reducing agent, usually hydroquinone, it forms black elementary silver.
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Discovery Purple chromogen
The use of Discovery Purple chromogen and other innovative chromogens, such as Ventana Purple, provides excellent contrast that is easily differentiated from natural tissue pigmentation. This is particularly advantageous when analysing tissues with natural pigmentation, such as endogenous melanin protein, where traditional chromogens like DAB (3,3'-Diaminobenzidine) may be difficult to differentiate.
The selection of chromogen colours, such as Discovery Purple, should be based on target biomarker expression patterns, levels, and antibody performance details. Lower-expressing targets can be highlighted with naturally more visible colours, such as purple, to enhance visibility when viewed by eye. Additionally, the availability of a wide variety of colours, including Discovery Purple, provides limitless opportunities to optimize assay performance in multiplexing applications.
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Chromogenic IHC
The chromogenic dye contains a tyramine group that becomes reactive upon interaction with HRP and hydrogen peroxide, forming a highly reactive intermediate. Nearby tyrosine residues in endogenous proteins also become activated and condense with the dye intermediate, resulting in the formation of a covalent bond and local deposition of the chromogenic dye. Finally, the sample is counterstained with a nuclear stain, such as DAPI or hematoxylin, to visualize the cellular structures under a microscope.
Traditional chromogens like 3,3'-Diaminobenzidine (DAB) and Fast Red have broad absorption spectra and produce a robust color with a dynamic range. DAB is the most widely used chromogen in IHC due to its stability and ability to provide a nice contrast with Hematoxylin counterstains. However, it may be difficult to differentiate DAB from natural tissue pigmentation in some cases.
To address this issue, researchers have developed innovative chromogens, such as Purple, Green, Yellow, Blue, and Teal, which provide excellent contrast and can be easily differentiated from tissue pigmentation. These new chromogens are based on fluorophores, allowing for unique color generation and narrow-range light absorption, making them highly compatible for IHC multiplexing.
Silver staining chromogenic IHC is another technique that utilizes the chemical reduction of silver ions to metallic silver. This process forms an insoluble black precipitate at the site of the primary and secondary antibody complex, providing strong contrast and high sensitivity.
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Frequently asked questions
Chromogens are dyes that are designed with a tyramine group that becomes reactive after interacting with HRP in the presence of hydrogen peroxide to form a highly reactive intermediate.
3,3’ diaminobenzidine produces a dark brown precipitate that is insoluble in alcohol. Nitro-blue tetrazolium/5-bromo-4-chloro-3’-indolyphosphate (NBT/BCIP) produces an intense, insoluble black-purple precipitate.
Chromogens are used to achieve improved target biomarker detection. They can also be used to build up a composite image of multiple antigens via cycles of primary antibody staining, indirect detection with HRP-conjugated secondary antibodies, imaging, and erasing.
