Choosing the Best Substrate

Choosing the Best Detection Module for IHC

Immunohistochemistry (IHC) uses antibodies to selectively detect proteins in a cell or tissue. Detection relies on a secondary antibody to detect the first. This antibody will be conjugated to a reporter. The most common reporter molecules you will encounter in IHC are alkaline phosphatase and horseradish peroxidase. Both of these proteins react with target substrates to form a colored product.

How do you make the right choice of detection module? This will depend heavily on the endogenous components of the cells on the slide. For example, hematopathology specimens have a high natural level of peroxidases. Therefore, you would want to use alkaline phosphatase to reduce non-specific staining. Conversely, certain immune cells like macrophages have a high level of acid phosphatase. As you can tell, horseradish peroxidase would be the best protein to choose.


You must also consider the types of preservatives you are including in your buffers. Sodium azide is a common anti-microbial substance included in many antibody preparations. Unfortunately, azide is a strong inhibitor of peroxidases.

Furthermore, you can consider the sensitivity of each detection medium. For a few reasons, alkaline phosphatase is the more sensitive of the two in general. Most importantly, the products of the horseradish peroxidase reaction inhibits the enzyme. This extinguishes the reaction.

Peroxidase is still more common since it can be used with more vibrant counterstains than with phosphatase. Slices treated with alkaline phosphatase also cannot be permanently mounted to a slide. Diffusion of the product can also be a problem if your slice happens to be thick.

In terms of amplification, tyramide-based methods allow for the amplification of signal from horseradish peroxidase. The ability to achieve better contrast against a nuclear stain enables a better resolution of peroxidase detection methods. You definitely want to be careful with these amplification procedures. They take quite a long time to process, and you will not know the results until you are finished. If the signal is too strong, it will be very difficult to correct later.

Overall, the ease of use that defines horseradish peroxidase makes it the most common choice for detection module in IHC. Certain cells cannot use it, but you will find that most systems are amenable to peroxidase-based methods. Substrate kits, as well as enhancers to increase immunoreactivity, are commercially available.

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