Enzyme

IHC detection systems usually include two different enzymes—horseradish peroxidase (HRP) and alkaline (AP)—and this is the first factor to consider when selecting an IHC substrate. HRP-based detection systems generate sharp and dense precipitates. Applications that require detection of intracellular targets or highly delineated locations frequently benefit from the use of HRP-based detection systems. Conversely, AP-based detection systems are the preferred choice for applications that require visualization of underlying tissue structure, as their precipitates are diffuse and translucent.

HRP substrates produce sharp and dense reaction products; AP substrates yield a more diffuse and translucent precipitate.

You should also be aware that substrates are enzyme-specific; an HRP substrate will not react with an AP-based detection system and vice versa. This specificity brings additional flexibility to your experiments as it allows for multiple staining applications and may help circumvent problems with endogenous enzyme activity by simply changing the enzyme detection system.

Color

When choosing the substrate color, the first factor to consider is the need for contrast between the target and the tissue specimen. Some tissues, such as melanoma, are naturally pigmented or have brown or black tissue deposits. Using a substrate that has an excellent contrast is imperative to avoid false-positive interpretations in these situations. You should also consider the color of any counterstain that will be used.

Contrast is also essential when doing double-staining. For example, if you select a red substrate for one antigen, you may want to choose a blue substrate for the other target. However, be aware that some substrates, despite their contrasting colors, are simply not compatible or must be applied in a specific order in the multiplex workflow—Vector Blue is compatible with Vector Red when Vector Blue is used for the first antigen label.

For a complete guide to compatibility between enzyme substrates, please refer to our IHC Multiplexing Guide.

Sensitivity

Different substrates vary in their sensitivity, which offers additional flexibility and control when designing experiments. Higher sensitivity allows better detection of low abundance proteins. Different formulations of the same substrate also have varying sensitivity. For maximum sensitivity, ImmPACT substrates should be chosen over other substrate formulations offer by Vector Laboratories.

Using high-sensitivity substrates may result in reduced cost over time

Choosing enzyme substrates with high sensitivity also has the potential to reduce costs in the long run. The figure below shows that an eight fold increase in the primary antibody dilution still yields an appropriate staining intensity. Some substrates have the added benefit of increased sensitivity with longer incubation times. The AP substrate BCIP/NBT, for example, offers an incubation time of up to 24 hours.