Immunohistochemistry (IHC) Resources

slanted slides for IHC resources
Flexible Immunohistochemistry (IHC) capabilities to support research and development of cancer diagnostics

Immunohistochemistry (IHC) is a technique that uses antibodies applied to tissues to detect targets of interest–usually a specific protein (antigen). It is performed on thinly sliced formalin-fixed paraffin embedded (FFPE) tissue mounted on slides and interpreted using a microscope.

The technique is used to diagnose disease, measure response to therapeutic drugs, and research countless basic biological applications. It is an essential tool for cancer research and diagnostics. IHC can identify cell types and provide information on dysregulated biochemical pathways.

In the last decade, IHC has enabled several important companion diagnostics. In 1998, Genentech (a Roche company) developed the first companion diagnostic test. The use of primary antibody clone 4B5 on breast cancer tissue can identify tumors caused by multiple copies of the HER2 gene, qualifying individuals for treatment with the highly specific anti-cancer drug Herceptin.1

How IHC works

There are multiple ways to perform immunohistochemistry, but the following components are often involved:

The protein target is commonly referred to as the antigen.  An antigen is the protein or peptide to which the antibody has been raised. IHC locates the protein targets in the tissue with the primary antibody, and detects it with a variety of chemistries.

The primary antibody is an antibody that binds tightly to a specific protein target/antigen.  Many antibodies are approved for use in the diagnosis of diseases such as  lung cancer, breast cancer, cervical cancercolorectal cancer, dermatological cancer, hematopathology cancer, prostate cancer and other solid tumors.  Primary antibodies can be labeled directly with a marker, but for greater sensitivity they are often used with a secondary antibody.

The secondary antibody recognizes the primary antibody. Use of a secondary antibody can result in greater sensitivity since it is coupled with an enzyme  that reacts with chromogens or fluorescent molecules, causing them to be deposited on the tissue.  To learn more about the use of secondary antibodies in IHC, see the Educational Resources.

An infographic that walks though how immunohistochemistry works as a technique to detect cancer.

Two notable enzymes in IHC

Horseradish peroxidase (HRP) and alkaline peroxide (AP) are two enzymes commonly used in IHC and often are coupled to secondary antibodies. HRP is a 44-kDa protein that activates in the presence of hydrogen peroxidase (H2O2) and catalyzes the oxidation of substrates. Activated HRP oxidizes electron donors, which then reacts with electron-rich aromatic compounds, such as 3.3-diaminobenzidine (DAB) to produce an insoluble colored product bound to the tissue.

Alkaline phosphatase (AP) is an 86 kDa protein enzyme that reacts with its substrates to hydrolyze them into phenolic compounds and phosphates. These phenolic compounds then interact with colorless diazonium salts and yield a colored product (chromogen). Fast Red, Fast Blue, or NBT/BCIP are commonly used chromogens with AP in IHC.

Visualizing antigen-antibody interaction with chromogens and fluorescence

While the primary antibody can bind to the protein target, it will not be visible without either the use of a fluorescent or chromogenic molecule. Historically, staining has relied upon the use of peroxidase and alkaline phosphatase enzymes to catalyze conversion of traditional chromogenic stains, such as 3.3-diaminobenzidine (DAB), Fast Red, and Fast Blue, into insoluble products on the tissue.

Several new and unique chromogens have been developed by the Ventana Research and Development Team to greatly extend chromogenic IHC capabilities beyond traditional chromogens. These next-generation chromogens can be deposited through covalent bonds rather than precipitation for better stability. Some new chromogens have translucent qualities enabling co-localization studies when multiplexed

A chromogen stained slide using IHC
A fluoerescence stained slide using immunohistochemistry

Visit the Chromogen Resources Page
for more information

What is multiplexing?

The ability to detect multiple targets in a single tissue sample, known as multiplexing, can be accomplished using multiple primary antibodies for IHC, or multiple probes for ISH. IHC and ISH can even be multiplexed together on the same slide. The use of translucent chromogens can result in the formation of additional colors when they stain the same subcellular component. 


Why is multiplexing important?

Multiplexing enables researchers to gain more information from a single tissue sample.  By investigating multiple biomarkers simultaneously, multiplexing generates unique information that is not possible to gain from single stains.

It allows investigators to do the types of experiments that are difficult to do using serial sections, such as protein co-localization, and determining the spatial relationships of different cell types.

A graphic depicting multiplexing on a slide

Visit the Multiplexing Resources Page
for more information


DISCOVERY ULTRA research instrument for IHC/ISH

The DISCOVERY ULTRA research instrument is the solution for scientists and research professionals who demand more than what conventional immunohistochemistry (IHC) and in situ hybridization (ISH) research methods have to offer.


Automated, High-Flexibility IHC/ISH Slide Staining for Assay Development

  • The DISCOVERY ULTRA system has 30 independent slide drawers that enable you to run 30 different staining protocols simultaneously.
  • Fully flexible software allows the addition of manual touch points at any step for even greater flexibility — no locked drawers!
  • The system provides the ability to fully automate a broad range of IHC and ISH assays, including FISH, gene and protein ISH/IHC, mRNA ISH and multiplexed assays (with any combination of IHC and ISH).


  1. U.S. Food and Drug Administration. List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools). Accessed June 28, 2022.

DISCOVERY instruments and reagents are for research use only. Not for diagnostic purposes.

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