Immunohistochemistry & IHC Stainers Explained

Scientists and clinicians routinely search for abnormal features that indicate disease. Changes in how cells and tissues appear are one such indicator. Researchers can also look for changes in biomolecule concentrations within clinical specimens. 

In both cases, scientists use tools that help monitor granular appearance changes that are associated with disease.

Immunohistochemistry (IHC) represents one such powerful tool. Scientists use IHCs to stain cells and view them underneath a microscope. This approach allows them to identify and develop biomarkers of disease status and patient responses. The secret to this success lies in harnessing the immune system to observe specific cellular features with a microscope. 

Nonetheless, IHCs require a reproducible protocol from tissue fixation to antibody labeling. IHC autostainers automate the staining process, increasing the reproducibility of IHC protocols. A wide range of IHC autostainers exist, each with unique features that can help you discover and evaluate distinct disease biomarkers.

What Is IHC?

IHCs are assays that focus on detecting specific biomolecules using the binding activities of antibodies. Antibodies are a key component of your body’s immune system. It allows your body to detect foreign substances called antigens so your immune cells can deal with them accordingly. All antibodies contain a core region that acts as the antibody’s foundation. 

However, the success of IHCs depends on an antibody’s variable regions. These regions bind to specific antigens through chemical bonds, including electrostatic interactions and hydrogen bonds.

 Scientists have access to two primary approaches for conducting an IHC. Both approaches produce a detectable visual signal when viewed under a microscope.

• Chromogenic immunohistochemistry (CIH): In CIH, the antibodies that bind to their antigens have enzymes attached to the core region. Horseradish peroxidase and alkaline phosphatase are the most common enzymes employed, both of which have also been used in immunoassays. Both assays cause specimens to stain a blue color after samples are incubated with the antibodies and their respective substrates. These substrates are called chromogens because they emit a blue color after their respective reactions are completed.
• Immunofluorescence (IF): In an IF assay, the antibodies employed are attached to a fluorescent dye instead of an enzyme. After the antibody binds to its antigen, a light source excites the dyes, emitting light through the photoelectric effect. The assays not needing a chemical reaction affords IFs increased sensitivity and specificity when taking microscopic images. IFs can also be used to sort cell populations based on specific characteristics using flow cytometry.

IHC Applications

IHC holds an important place in medical practice. IHCs have helped clinicians diagnose disease for almost a century. Its success stems from its ability to stain and highlight morphological and molecular features in cells that are associated with many cancers, including:

• Neoplasms: More commonly known as a tumor, neoplasms are abnormal masses of tissue that may be benign or cancerous. IHC allows researchers to determine the origins of tumor tissue or assess its behavior and growth. For instance, researchers can stain smooth muscle actin fibers to distinguish muscle tumors from other kinds of intestinal tumors.  
• Lung cancer: IHCs can also be used to identify and develop molecular biomarkers of cancer that can be visualized underneath a microscope. In lung cancers, adenocarcinomas – the most common kind of lung cancer – can be diagnosed by staining cells for thyroid transcription factor 1 (TTF1). Squamous cell lung carcinomas can also be identified by staining a different biomarker called p40. 

IHCs have also been used to diagnose infectious diseases, such as:

• Syphilis: Scientists have developed antibodies that bind to an avidin-biotin peroxidase complex to detect Treponema pallidum, the causative agent of syphilis. With this technique, specific epitopes for the microbe are exposed before the samples are stained with antibodies that target the microbe.
• Bacteremia: Researchers have also developed IHCs to diagnose bacteremia from cat scratches. Here, they used IHC to determine that people who had healthy immune systems could also be exposed to Bartonella henselae and experience bacteremia.

How IHC Staining Takes Place

Ensuring that your IHC experiments yield robust data requires a good understanding of a typical IHC workflow. This pipeline is divided into four major steps:

• Fixation: The first step in any IHC assay preserves tissues in place, preventing them from decaying. Scientists have access to many kinds of fixatives, each having distinct mechanisms that affect the fixation protocol and the quality of your IHC images. The two most common classes of fixatives are:

• Formalin (or formaldehyde dissolved in water) is the most common fixative, fixing cells by cross-linking soluble proteins to the cytoskeleton with covalent chemical bonds. Think of a molecular anchor that holds the cells’ proteins in place. Embedding of the samples with paraffin usually follows, holding small, fragile tissues in place for sectioning without losing the tissues. Although formalin fixation preserves protein tissues well, a longer fixation time can mask proteins from antibodies, reducing staining efficiency.
• Alcohols are the other fixative, with methanol and ethanol being the most common. Researchers don’t have to be concerned about antigen masking with alcohol fixation, but this fixation method increases the risk of protein degradation. Degrading proteins increases the risk of modifying epitopes, reducing antibody binding to their antigens.

• Antigen retrieval: This step applies exclusively to formalin-fixed, paraffin-embedded tissues. Formalin fixation increases the chance of antibodies failing to bind to their antigens. This step alleviates this risk by unmasking the cross-linked proteins. The process involves boiling fixed tissues in water to improve antibody binding, ultimately enhancing the IHC signal.
• Blocking: Antibodies can bind to biomolecules other than their intended antigen targets, producing noise in IHC images. Whereas antigen retrieval unmasks their targets, blocking prevents antibodies from binding to non-target molecules. The process involves using a biomolecule to prevent certain cellular components from being available for binding. Multiple blocking reagents can be used, such as protein-blocking reagents for specific proteins, avidin to block biotin from being available for binding, or hydrogen peroxide to block peroxidases.
• Antibody labeling and visualization: After the samples have been processed, they are ready for staining. Here, samples are first labeled with a primary antibody designed to bind to the specific biomolecules you’re interested in. Then, you have the option of adding secondary antibodies that bind to the primary antibodies (insert link to immunoassay article). Deciding whether to add secondary antibodies for your IHCs depends on multiple variables:

• Level of signal strength desired: Secondary antibodies provide greater signal strength in IHC images. The increased signal strength increases the IHC’s sensitivity by allowing multiple secondary antibodies to bind to the same primary antibody. Secondary antibodies are most useful if your desired targets are lowly abundant in your samples.
• Signal specificity: If you want to reduce the risk of background noise, using only primary antibodies may be the way to go. Secondary antibodies increase the risk of cross-reactivity, occurring when antibodies bind to unintended targets. Secondary antibodies’ potential off-target binding activity increases the risk of image noise within your IHC images. 

Before you begin any IHC experiment, you should take steps to optimize each of these steps. Doing so will help you produce visual images that will best help you study and diagnose disease.

IHC Autostainers

Once you have decided on a robust IHC pipeline, finding an autostainer comes next. IHC protocols involve a multistep process that can make producing reproducible images underneath a microscope difficult. 

Autostainers automate each of these steps, improving cost and time efficiency with a reproducible protocol in place. Here below are just some of the IHC autostainers available on the market:

• Autostainer Link 48 (Agilent): This autostainer allows 48 slides to be processed in less than 3 hours, enabling high-throughput IHC. With this autostainer, each step of the IHC pipeline is automated, from mounting slides to adding coverslips for viewing. The autostainer can also be paired with the PT Link and DakoLink software. The former mediates the fixation and target retrievals, while the latter facilitates IHC automation.
• BOND Automated IHC Staining System (Leica): This autostainer is well-suited for high-throughput IHC as well, allowing up to 30 slides to be stained in a 2.5-hour run. The system also comes with the BOND Polymer Refine Detection system that contains a series of reagents to increase antigen retrieval and improve blocking efficiency.
• ONCORE PRO Staining Platform (Biocare Medical): This automated slide staining system works with up to 36 slides in a single IHC run. The system conducts every step of the staining procedure, from fixation to antibody detection. The ONCORE PRO also has unique reaction modules that encase slides between a heated platform and a novel containment chamber. This feature reduces reagent usage and allows for gentle agitation, producing maximal staining and minimal background noise.
• BenchMark ULTRA (Roche): This autostainer allows up to 30 samples to be stained simultaneously in an IHC assay. Roche also provides a wide-ranging menu of 250 ready-to-use IHC assays validated for studying mouse and rabbit models. The BenchMark ULTRA also allows researchers to generate custom time and temperature protocols to suit any kind of IHC experiment.

Excedr’s Pricing Model

At Excedr, we provide a unique pricing model for our leasing programs. Our leases offer a way to procure  the autostainers you need and produce data that suits your experimental and logistical needs. 

For us to best meet your needs, you should consider a myriad of factors that affect the kinds of autostainers you use to answer your research questions.

• What kinds of samples do you want to work with? IHCs are typically performed on clinical specimens. Each specimen contains cells that have unique expression patterns, whether healthy or diseased. Each sample type also contains a diverse array of biomolecules that affect staining efficiency. Knowing the characteristics of your samples will help you determine which autostainers can enable you to process samples with the protocols you design.
• How many samples are you processing? Each autostainer can process a similar number of samples for IHC experiments. However, knowing the number of samples you will process daily will help you select the autostainer that best suits your research and diagnostic needs.
• Are you studying new or established biomarkers? Some autostainers come with antibody menus that allow you to conduct reproducible experiments on a set of validated IHC assays. Other autostainers are compatible with other pieces of equipment that afford additional capabilities, such as automated antigen staining. Knowing what kinds of biomarkers you are examining will help you identify the autostainers best suited to help you assess disease and health biomarkers under a microscope. 
• How do companies distribute their equipment and reagents? Some companies provide unlimited access to equipment provided you exclusively use their consumables. Other companies may provide their own programs depending on the sets of reagents compatible with their equipment. From this, consider whether you plan to use your own reagents or to conduct your IHCs with established pipelines and biomarkers.

Speak with Us Today

Reproducibility holds a vital place in IHC studies. Autostainers provide the means to automate IHC staining protocols and ensure reproducibility. Generating high-quality, reproducible images allows researchers to assess disease states and monitor patient responses to therapy at the cellular and molecular levels. 

Take a big step in disease diagnostics and research with our array of IHC autostainers. We boast autostainers from many brands, allowing hundreds of specimens to be stained daily. Whatever your IHC needs, we can help you meet them. 

Since we don’t carry an inventory, you can freely consider what autostainer works for you and have the instrument ready for your lab. Read through this article as a resource and then speak with a representative to see how we can help you select an autostainer that will help you meet your research needs. Are you interested in leasing an autostainer? Let us know!