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Educational Resources for IHC, ISH and Multiplexing

ihc gastric slide image

Find additional resources about immunohistochemistry and in situ hybridisation

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

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Related resources

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 cancer, colorectal 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

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).
Learn more

Frequently Asked Questions

Yes. Roche carries a broad line of ready-to-use research products. The DISCOVERY ULTRA is flexible to run any third party reagents as well. These reagents include but are not limited to blockers, primary antibodies, detection antibodies, and detection kits. Reagents can be manually applied or fully automated.

Yes. Roche currently does not have a ready to use kit for detecting cell death, but third party assays such as TUNEL can be automated on the DISCOVERY ULTRA.

Yes, however Roche does not offer directly labeled fluorescent probes. Researchers can obtain superior results using our broad library of ready-to-use DISCOVERY fluorescence kits.  These kits use Tyramide Signal Amplification (TSA) for superior signal, and can be used with DIG- and DNP-labeled probes. Additionally, key steps are incorporated in the DISCOVERY Universal Procedure Software to allow directly labeled probe hybridisation.

Any tissue type or species can be run on the DISCOVERY ULTRA, as long as the appropriate antibodies are incorporated into the assay. Note that the Omnimap and Ultramap HRP-conjugated antibodies are available as single species to detect mouse, rabbit, rat, and goat antibodies for more flexibility in designing assays.

DISCOVERY Universal Procedure Software has multiple opportunities to add blocking steps. Key steps include Option steps and Antibody blocking steps. Note that the Option steps will allow blocking reagents to incubate then they will be rinsed off. Antibody Blocking step will incubate then the following antibody will co-incubate with the blocking reagent. The two application selections allow for maximum flexibility in incorporating the blocking reagents.

DISCOVERY HQ products use unique haptens for robust sensitivity. In addition to HQ detection, DISCOVERY amplification kits use both unique haptens and tyramide for enzymatic amplification. The amplification kits can boost signal while maintaining specificity. See DISCOVERY Detection for more details.

Yes. Ready-to-use reagents can be registered and used immediately. Third party reagents can be either applied manually or fully automated in a user fillable dispenser. Protocols with manual applications will beep when the reagents need to be applied, allowing the user to manually access the slide to manually pipette on reagents.

DISCOVERY Purple, DISCOVERY Yellow, and DISCOVERY Teal are the optimal chromogens for detecting co-localisation. DISCOVERY Purple and DISCOVERY Yellow will result in a reddish/orange hue. DISCOVERY Purple and DISCOVERY Teal will result in an indigo blue hue. DISCOVERY Yellow and DISCOVERY Teal will result in a green hue.

The amount of chromogenic colours that can be combined depends on many variables, including the stability of epitopes being detected, and the compatibility of chromogens being used. Conditions for each target should be optimized individually, then combined and evaluated for equivalent performance.

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

Reference:

  1. U.S. Food and Drug Administration. List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools).  https://www.fda.gov/medical-devices/in-vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-in-vitro-and-imaging-tools. Accessed June 28, 2022.