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
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.
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.
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
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.
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.
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.
Reference:
Chromogen Product information
View Full TableChromogen Product information
| Chemistry | Chromogen | Roche Number |
|---|---|---|
| Horseradish Peroxidase | DISCOVERY Red | 9576819001 |
| DISCOVERY Yellow | 8502641001 | |
| DISCOVERY Blue | 9582690001 | |
| DISCOVERY Purple | 7053983001 | |
| DISCOVERY Silver | 7053649001 | |
| DISCOVERY Teal | 8254338001 | |
| DISCOVERY Green | 8478295001 | |
| ChromoMap DAB | 5266645001 | |
| Alkaline Phosphatase | DISCOVERY Yellow | 7698445001 |
| DISCOVERY Red | 7425333001 | |
| ChromoMap Blue | 5266661001 | |
| ChromoMap Red | 5266653001 |
Detection Product information
View Full TableDetection 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 |
| 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 |
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.
The advent of microprocessor-controlled robotics enabled the automation of previously manual tasks, and freed laboratory technicians to make more valuable use of their time.
A pioneer in IHC automation, Ventana launched the “ES” automated stainer in 1988. Since that time, customer feedback and innovative engineering have combined to continually improve the sophistication, reliability, and high-quality staining performance of the BenchMark and DISCOVERY ULTRA instrument platforms. Today, the DISCOVERY ULTRA instrument and Universal Procedure Software lead the way in the research and development environments of leading pharmaceutical companies, Contract Research Organizations (CRO) and Academic Medical Centers.
Similarly, the closely related BenchMark ULTRA is the leading clinical IVD automated staining platform in the world. Every minute, 95 patient samples—more than 50 million a year—are stained on BenchMark IHC systems around the world, providing consistent, reproducible quality.
IHC and ISH experiments can be expensive and time consuming. Automation of IHC and ISH brings several advantages over manual staining on the bench.
Software that runs automated staining instruments for clinical diagnostics is limited in protocol design for the purposes of clinical safety. Software for a research instrument can be far more open and flexible than that of a clinical instrument because it is not under the constraints of regulations that apply to the FDA-approved clinical IVD testing environment.
After more than a decade of listening to and collaborating with our Research customers, Roche DISCOVERY Universal Procedure Software has evolved to have unparalleled flexibility, and enables researchers to perform multiplexing experiments of high complexity with both chromogenic and fluorescent assays.
There are two choices in automated staining workflows: batched drawers and individual staining drawers. Batched drawers contain multiple slides, but this design results in a number of constraints that negatively impact lab workflow and experimental flexibility. For example, in a batched system, the protocols for each slide must all employ the same temperature. Further, batched drawers must remain locked until the longest protocol is finished, and this reduces workflow efficiency.
A comparative workflow analysis for automated chromogenic immunohistochemistry (IHC) and in situ hybridization (ISH) staining
Overview: Continuous random access to independent, single-slide reaction chambers maximizes protocol flexibility, allowing technicians to shape the platform to meet varying project requirements while enabling laboratories to operate at full instrument capacity at all times.
Individual slide drawers allow instrument operation at maximum capacity by running ISH samples alongside IHC samples, long and short protocols, and different detection chemistries simultaneously. Unlike batch processing, when the slide is ready, it can be immediately removed and coverslipped.
Individual slide drawers enable labs to run a higher number of different protocols simultaneously.
With batched staining workflow, lab personnel are required to take time out to figure out the best way to group slides for staining, which lowers productivity. This laborious step is eliminated by the use of individual staining drawers.
With individual slide drawers, open drawers can be immediately started without disrupting other runs or overall productivity.
Unlike batched drawers locked to one temperature, individual slide drawers have completely independent temperature control—slide by slide.
With the Roche Universal Protocol software, manual touchpoints can be placed virtually anywhere within protocols enabling you to do whatever crazy thing you’d like to do at any time during the run! This is not something that can be done with batched drawers that remain locked during the duration of the longest staining protocol in the drawer.
The DISCOVERY ULTRA platform is completely open to the use of reagents of your choice and sources. This flexibility is enabled by user-fillable dispensers and Universal Protocol software.
Roche researchers have developed sophisticated detection chemistries and unique chromogens that are specifically optimized for IHC, ISH, and multiplexing.
For more information, visit these resource pages:
VENTANA Connect software solution links your existing Laboratory Information System (LIS) with Roche Tissue Diagnostics portfolio of diagnostic instruments, and enables the LIS to send orders and receive status back from Roche Tissue Diagnostics stainers using the standard HL7 protocol.
Possible uses for Ventana Connect software solution include:
Connect customers’ LIS software through a single middleware application.
Receive order information from the customers’ LIS software and route this to appropriate products.
Route data/information (status updates, reports, analysis data, etc.) back to the customers' LIS software.
See how our partnerships with leading biotech organizations help create cutting-edge solutions for your lab.
3rd party solution for LIS integration
Access a learning management system that features training content on-demand for all lab personnel and knowledge levels.
Learn more
Link to key peer-reviewed publications for the latest findings on use of IHC in research and diagnostics.
See videos on topics related to IHC and its use in diagnostics and research.
Download pdfs from our library of white papers and brochures pertaining to IHC and related topics.
DISCOVERY instruments and reagents are for research use only. Not for diagnostic purposes.
