Traditional IHC chromogens make the presence of target biomarkers visible by light microscopy. An enzymatic reaction catalyzes the change of a soluble chromogen compound into an insoluble colored precipitate that binds to the tissue at the site of the biomarker target. Traditional chromogens produce a dark, opaque signal.
Typically driven by horseradish peroxidase (HRP) or alkaline phosphatase (AP), chromogens traditionally used in IHC and ISH detection include:
DAB –3,3'-diaminobenzidine – robust color, dynamic range, highly stable, permanent, insoluble in water and alcohol. DAB staining is the most widely-used chromogen in IHC.
For chromogenic IHC, the deposition of DAB chromogen onto a tissue section on a microscope slide involves an enzymatic reaction that is commonly used in IHC.
First, a primary antibody binds to a specific target protein in the tissue section, and a secondary antibody linked to an enzyme, usually horseradish peroxidase (HRP), binds to the primary antibody. When the HRP-labeled secondary antibody comes in contact with a solution containing the DAB chromogen and hydrogen peroxide, the following chemical reaction occurs:
HRP + H2O2 + DAB → DAB•+ + H2O + oxidized HRP
In this reaction, HRP acts as a catalyst and oxidizes DAB in the presence of hydrogen peroxide to generate a brown insoluble product that deposits on the tissue section. The DAB•+ product is a highly reactive intermediate that polymerizes to form a dark brown precipitate at the site of the protein of interest. This deposit can be seen under a microscope, allowing the location and distribution of the protein to be determined.
Fast Red-based chromogens – high contrast color, prone to fading and/or blushing when exposed to alcohol or xylene.
Silver Stain – strong contrast, high sensitivity.
Silver staining chromogenic IHC works by the chemical reduction of silver ions to metallic silver, which forms a visible, insoluble precipitate at the site of the primary and secondary antibody complex. The chemical reduction of silver ions to insoluble metallic silver precipitate occurs through a series of redox reactions. The reducing agent used in the silver enhancement step is usually a mixture of formaldehyde and citric acid. The formaldehyde reacts with the silver ions to form a complex that is then reduced by citric acid, and produces insoluble metallic silver precipitate that appears black through the microscope.
The Research and Development Team at Ventana have developed several new chromogens to expand ISH and IHC multiplexing capabilities in brightfield microscopy. These new chromogens are based on fluorophores to allow for unique color generation and very narrow-range light absorption to improve compatibility for ISH and IHC multiplexing. By basing IHC/ISH chromogen chemistry on fluorophores, Ventana researchers have generated new, narrow-absorbance colors such as DISCOVERY HRP-driven Purple, Red, Yellow, Blue, Green and Teal. Tyramide chemistry is used to covalently deposit these unique chromogens for excellent stability.
For more information, see this peer-reviewed publication:
Tyramide chemistry is a powerful technique that can be used to stain or even amplify the signal of immunohistochemistry (IHC) staining. One major advantage is that the dye forms a covalent bond with proteins in the tissue for greater stability.
Like traditional IHC, the primary and secondary (HRP) antibodies are incubated with the tissue sample to bind to the antigen of interest. The chromogenic dye is designed with a tyramine group that becomes reactive after interaction with HRP in the presence of hydrogen peroxide to form a highly reactive intermediate. Similarly, tyrosine residues in the endogenous proteins nearby will become activated after contact with the HRP, and then condense with the dye intermediate to form a covalent bond and local deposition of the chromogenic (or fluorescent) dye. Finally, the sample is counterstained with a nuclear stain, such as DAPI or hematoxylin, to visualize the cellular structures. The result is a highly specific and sensitive signal.
| Chemistry | Chromogen | Roche Number |
|---|---|---|
| Horseradish Peroxidase | DISCOVERY Red | 09576819001 |
| DISCOVERY Yellow | 08502641001 | |
| DISCOVERY Blue | 09582690001 | |
| DISCOVERY Purple | 07053983001 | |
| DISCOVERY Silver | 07053649001 | |
| DISCOVERY Teal | 08254338001 | |
| DISCOVERY Green | 08478295001 | |
| ChromoMap DAB | 05266645001 |
| Chemistry | Chromogen | Roche Number |
|---|---|---|
| Alkaline Phosphatase | DISCOVERY Yellow | 07698445001 |
| DISCOVERY Red | 07425333001 | |
| ChromoMap Blue | 05266661001 | |
| ChromoMap Red | 05266653001 |
A wider choice of chromogenic colors gives researchers an increased ability to customize IHC and ISH assays to achieve improved target biomarker detection. Traditionally, DAB has been the main chromogen of choice for general applications with Red following close behind for tissues with pigmentation. DAB provides a nice contrast to Hematoxylin counterstains and allows even low expressing targets to be easily visualized. However, if the tissue being analyzed has natural pigmentation, such as endogenous melanin protein, DAB can be hard to differentiate from the melanin in the tissue. Similar to Red chromogen, the innovative Ventana chromogens such as Purple, Green, Yellow, Blue and Teal can provide excellent contrast that is easily differentiated from natural tissue pigmentation.
When multiplexing IHC or ISH, having a wide variety of colors to choose from can provide limitless opportunities to optimize assay performance. Selection of colors should be determined by target biomarker expression patterns and levels as well as antibody performance details. Lower expressing targets can be highlighted with colors that are naturally more visible when viewed by eye such as Red, Purple, DAB, and Silver. Yellow, Teal, Blue, and Green are useful for general cell markers to complement target analysis and provide a colorful and informative depiction of the tissue environment.
This tool demonstrates some of the applications available on the Ventana DISCOVERY ULTRA, and gives you the ability to create and save a variety of assay types and generate associated product lists.
If the biomarkers of interest are present in the same sub-cellular compartment (nucleus, cytoplasm or cell membrane) this overlapping expression of targets is a key consideration when selecting chromogen options for IHC multiplexing. When designing a chromogenic IHC assay that includes overlapping target biomarker expression, opaque colors such as DAB and Silver should not be used.
Traditionally, analysis of overlapping targets has been accomplished using immunofluorescence, but the availability of new Ventana translucent chromogens has provided an alternative method - in brightfield! Translucent chromogens allow a color shift when both colors are present in the same cell and sub-cellular compartment.
Availability of translucent chromogens such as Purple, Yellow, and Teal has opened up the ability to visualize overlapping targets in brightfield IHC or ISH multiplexed assays.
Visit our Multiplexing Resources page for more in-depth information on performing multiplexed IHC.
Co-localized DISCOVERY Purple and DISCOVERY Yellow IHC chromogens result in a fiery red/orange colors shift. DISCOVERY Teal and DISCOVERY Purple chromogens combine to highlight an indigo blue to deep purple color. A leafy green can be observed with the combination of DISCOVERY Teal and DISCOVERY Yellow.
Traditional chromogens such as DAB and Fast Red-based chromogens have very broad absorption spectra (see Figure 1 - Chromogen Spectra). Chromogens that have the narrower absorption ranges (Purple, Yellow, Teal) can take on translucent qualities. This is because narrow absorption chromogens leave more absorption spectra for other dyes to occupy when they are deposited in the same physical space and then mixing of colors is observed.
The following dyes can exhibit translucent properties as they contain only one primary color from the (cyan, yellow, magenta, black) color space: Discovery Purple (magenta), Discovery AP Yellow (yellow), Discovery Teal (cyan).
High Contrast Chromogens are made of multiple light-absorbing compounds, and perform more like more traditional broadly absorbing dyes (e.g. DAB and silver stain). These chromogens span more space in the CYMK visual spectrum: Discovery Green (cyan + yellow), Discovery Red (yellow + magenta), Discovery Blue (magenta + cyan). Because the dual dyes are each made up of two of the three primary colors in CYMK color space, their ability to mix for translucent qualities is more limited. These chromogens should be considered high contrast dyes, where the difference in color between the dye and the counterstain or white light is more pronounced.
This brochure contains information on HRP and AP Multimers, hapten-based detection chemistry, and hapten-based amplification reagents that can be used for immunohistochemistry (IHC).
This brochure contains information on chromogens that can be used with DIG and DNP-label in situ hybridization (ISH) probes.
| Product Name | Roche Number |
|---|---|
| OmniMap HRP anti-Ms | 05269652001 |
| OmniMap HRP anti-Rb | 05269679001 |
| OmniMap HRP anti-Rt | 05891892001 |
| OmniMap HRP anti-Gt | 06607233001 |
| UltraMap HRP anti-Ms | 05269695001 |
| UltraMap HRP anti-Rb | 05269717001 |
| UltraMap HRP anti-Rt | 05891884001 |
| UltraMap HRP anti-Gt | 06607241001 |
| UltraMap AP anti-Ms | 05269687001 |
| UltraMap AP antiRb | 05269709001 |
| Rabbit anti-DNP | 05269768001 |
| Product Name | Roche Number |
|---|---|
| DISCOVERY anti-DNP AP | 07339739001 |
| DISCOVERY anti-DNP HRP | 07339712001 |
| DISCOVERY anti-DIG AP | 07256302001 |
| DISCOVERY anti-DIG HRP | 07256299001 |
| DISCOVERY Anti-Mouse HQ | 07017782001 |
| DISCOVERY Anti-Rabbit HQ | 07017812001 |
| DISCOVERY Anti-HQ HRP | 07017936001 |
| DISCOVERY Anti-Mouse NP | 07425309001 |
| DISCOVERY Anti-Rabbit NP | 07425317001 |
| DISCOVERY anti-NP AP | 07425325001 |
| DISCOVERY Amplification HRP Kit | 06527698001 |
| DISCOVERY Amplification AP Kit | 06211445001 |
| DISCOVERY Amplification anti-HQ HRP | 06442544001 |
| DISCOVERY Amplification anti-HQ AP | 06472311001 |
| DISCOVERY HQ Amplification kit | 06472320001 |
| DISCOVERY BF Amplification kit | 07529422001 |
| DISCOVERY Anti-BF HRP | 07529449001 |
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DISCOVERY instruments and reagents are for research use only. Not for diagnostic purposes.
