One-step, two-step, amplify: How to choose the right IHC detection system

Immunohistochemistry (IHC) can be a powerful tool in your arsenal of techniques for analyzing tissue. Chromogenic IHC results in high color contrast for data analysis, making it a great detection system that empowers the exploration of scientific complexities such as spatial relationships, intracellular signaling, or the tumor microenvironment. Detection systems such as IHC allow you to visualize your protein of interest by taking advantage of the high affinity of antibodies to their target antigens.  

The two types of detection systems are the one-step detection system and the two-step detection system: 

• One-step detection systems are good for single antigen staining, sequential double staining, and cross-reactivity reduction. These systems consist of micropolymers attached to highly cross-adsorbed, affinity-purified secondary antibodies that penetrate more deeply into thicker sections. These systems therefore provide specific primary antibody binding and avoid steric hindrance concerns. One-step systems also have a shorter assay time, so you get crisp images faster.  
• Two-step detection systems are good for low antigen expression levels and primary antibody dilution maximization. They consist of an amplifier antibody followed by an affinity-purified and cross-adsorbed polymer reagent to ensure high sensitivity and low background.  

Having different levels of sensitivity available for your IHC detection systems is like having a volume control for your signal but not for the noise. But how do you choose the perfect detection system? Will you use a one-step or two-step system? Do you need to multiplex antibody staining? Are you worried about endogenous biotin or enzyme activity that can obscure your signal? Before you panic, read on for a couple of key characteristics to consider when choosing a detection system. 

The general workflow of choosing a working set-up consists of selecting appropriate primary and secondary antibodies as well as a detection system. All these components work together like instruments in a band. Choosing each one carefully is important, but it is also essential to consider how each affects the entire experiment. Here are some considerations when selecting antibodies and detection systems. 

Choosing primary antibodies 

Primary antibodies should have high epitope specificity for the antigen of interest. In addition, the clonality needs to be appropriate for the experiment. For example, monoclonal antibodies bind a single epitope with high specificity and affinity. This can be very useful when detecting individual members of a protein family. However, changes in protein conformation and/or factors affecting epitope access can greatly impact the ability of monoclonal antibodies to bind. If you suspect these issues could affect your experiment, you might want to consider polyclonal antibodies, which can recognize multiple epitopes. Using polyclonal antibodies may lead to confounding staining though, especially if you’re looking to study a particular target that has closely related family members. A general rule of thumb: for the scarcest targets (least prevalent antigens) use the most sensitive detection reagents. 

Another consideration for primary antibody selection is the appropriateness for the fixation method. Some antibodies work only in certain experimental paradigms. For example, an antibody that recognizes a specific epitope might have access to that epitope in frozen-fixed tissue but might have a challenging time recognizing that epitope in formalin-fixed tissue, which can lead to crosslinking. If you’re using formalin fixation, you might want to consider incorporating an antigen retrieval protocol to unmask epitopes so your primary antibody can gain access. 

What about the species of your tissue? Have you tried using a mouse primary antibody on mouse tissue yielding results with almost zero color contrast? Talk about a nightmare scenario on precious tissue! Be sure that the host species of your primary antibody is different from the species of the tissue you’re investigating. However, if this is unavoidable in your experimental workflow, there are options available to you, such as Mouse on Mouse (M.O.M.^®) and Human on Human (H.O.H.^™) immunodetection kits, which turn up the signal and reduce the noise. 

Be sure to also choose the appropriate blocking reagents for your experiment. Background interference can come from a variety of sources including endogenous enzyme activity or interactions between detection reagents and tissue macromolecules. Choosing the correct blocking reagent reduces noise and boosts signal visibility. 

Choosing secondary antibodies and tertiary detection systems 

Like choosing your primary antibodies, secondary antibodies are key to ensuring excellent results. Secondary antibodies allow for signal amplification through a variety of methods (e.g., fluorescence or chromogenic) because multiple secondary antibodies can bind onto a single primary antibody. Secondary antibodies act much like primary antibodies and are raised against the species of the primary antibody. For example, if your primary antibody host species is rabbit, you should choose a secondary antibody that targets rabbit antibodies. When multiplexing your IHC, it’s good to have different species of primary antibodies so that your secondary antibodies can recognize each primary antibody and change them to different colors, offering high color contrast. After all, if you’re targeting multiple antigens using the same host species for the primary antibodies, using secondary antibodies targeting the primary host species will cause everything to look the same color under the microscope!  

Selecting the right substrates  

The two most widely used enzymes for chromogenic IHC are horseradish peroxidase (HRP) and alkaline phosphatase (AP). To choose the right enzyme substrate, there are four characteristics to keep in mind: sensitivity, color, visualization method, and heat resistance. Choosing an appropriate substrate that has sufficient sensitivity and high color contrast is especially essential when multiplexing antigen labelling or using counterstaining where multiple substrates might be necessary. Otherwise, when you look under the microscope you might be looking at insufficient levels of signal or at colors that are difficult to differentiate, making analysis tricky.  

Blocking endogenous activity 

Concerns about endogenous activity from enzymes or biotin can have a significant impact on what detection system you choose. Certain tissues have high endogenous peroxidase (e.g., spleen and kidney) and phosphatase (e.g., kidney, intestine, and liver) activity, which can lead to high background unless specific blocking reagents are used.  

Similarly, detectable levels of endogenous biotin may lead to high background noise. Therefore, using a non-biotin micropolymer-based detection system for your workflow could lead to enhanced signal with reduced background and superior access to epitopes. If this sounds like your situation, you have one- and two-step polymer-based detection systems to consider for your multiplexed chromogenic IHC, such as one-step ImmPRESS^® polymer systems or the two-step ImmPRESS^® amplified polymer system. 

Of course, if you’re not concerned with endogenous enzyme or biotin activity, you might want to consider two step avidin-biotin complex (ABC) detection systems instead. These systems are modular and versatile with high sensitivity and low background and are still one of the most widely used methods for staining. 

 Whether you’re a one-step or two-step sort of person, there are a variety of resources and kits to aid your workflow needs. For more tips and tricks, delve into our immunohistochemistry guide for more information.  

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