Immunohistochemistry (IHC) Resources

Chromogenic IHC and ISH Resources

IHC chromogenic slides for resources
What are the different types of chromogens used in IHC and ISH?

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:

Here’s how DAB staining works:

dab slide

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 slide

Fast Red-based chromogens – high contrast color, prone to fading and/or blushing when exposed to alcohol or xylene.

silver slide

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.

Roche’s next-generation chromogen technology

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.

  • Greater Control over staining intensity than traditional chromogens
  • Permanently Bound  HRP-driven tyramide deposition
  • Distinct, strong high-quality staining
  • Increases the Flexibility of multiplexing IHC and ISH
  • Greater Stability in EtOH and xylene than traditional chromogens
  • No Mixing! Ready-to-Use chromogens

For more information, see this peer-reviewed publication:  

Day, W., Lefever, M., Ochs, R. et al. Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ. Lab Invest 97, 104–113 (2017).

Here’s how Tyramide Chemistry staining works:

TSA DAB disposition

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.

HRP-driven chromogens

A sampling of HRP-driven chromogens for slide staining.
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

AP-driven chromogens

A samplig of AP-driven chromogens for slide staining.
Chemistry Chromogen Roche Number
Alkaline Phosphatase DISCOVERY Yellow 07698445001
DISCOVERY Red 07425333001
ChromoMap Blue 05266661001
ChromoMap Red 05266653001
How to select IHC / ISH chromogens that are best for your specific application.

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.

Use this helpful Detection Wheel to select the best reagents for your experiments!

Discovery Interactive Assay Design Tool screenshot
Use the Ventana Assay Design Tool to Design a Chromogenic IHC Assay

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.


Translucent chromogens enable multiplexed IHC co-localization.

translucent chromogens

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. 


How does using translucent chromogens for co-localization experiments result in a color change?

chromogenic absorbance

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.

Figure 1 - Chromogen Spectra

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 excellent for sensitivity and crisp detail.
high contrast chromogens

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.

Chromogenic resources

Brochure of Ventana chromogens for ISH and IHC

Read about our NEW HRP-driven Red, Yellow and Blue chromogens for IHC and ISH!

Don't live in the Stone Age with AP-Reds and HRP-DABs.  Explore the modern world of chromogenic multiplexing!

Don't live in the Stone Age with AP-Reds and HRP-DABs.  Explore the modern world of chromogenic multiplexing!

Image of brochure containing 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 HRP and AP Multimers, hapten-based detection chemistry, and hapten-based amplification reagents that can be used for immunohistochemistry (IHC).

Discovery Ultra product catalog
DISCOVERY product catalog
Fully automated IHC/ISH research
Downloand the brochure with information on chromogens that can be used with DIG and DNP-label in situ hybridization (ISH) probes

This brochure contains information on chromogens that can be used with DIG and DNP-label in situ hybridization (ISH) probes.

Detection Product information

View Full Table

Detection Product information

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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