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Top Considerations for Selecting Immunohistochemistry (IHC) Detection Reagents

Choice can be a double-edged sword—the freedom and access to a wide range of options to solve the exact problem you’re facing, coupled with what seems like endless possibilities and the challenge of finding the best approach to meet your needs. Faced with these choices you may start feeling overwhelmed.

When it comes to choosing your immunohistochemistry (IHC) detection system there are many considerations and important decisions to be made. Should you use a biotin or non-biotin system? How abundant is your protein of interest? What if your primary antibody species is the same as the tissue? Some decisions may seem inconsequential, but together they combine to give you the perfect picture and proof of your experimental success.

IHC is an established, sensitive method using antibodies to detect specific target antigens (proteins) in tissue sections. IHC exploits antibody-to-antigen affinity and utilizes enzymes for chromogenic reactions. If this is your first foray into IHC or you’re looking to improve your results, the choice of reagents and methods can be mind boggling. When designing your IHC assays, here are some vital considerations that will help you choose the right reagents:

Sensitivity – how abundant is the target antigen? If the target is easy to localize, you can employ less sensitive, more cost-effective methods. In fact, sensitivity may not be a key driving factor if your target is highly abundant. If it is, however, the intensity of staining achieved by both the detection systems and the chromogenic substrates needs to be carefully considered. Use the sensitivity of substates table below to choose a chromogen. Keep in mind the color and compatibility with other reagents (e.g., counterstains, mounting media, and other substrates if you’re multiplexing).

Background – nothing is more frustrating than finishing a multi-day staining experiment, looking under the microscope, and seeing what you think is signal, only to realize that you’re staring at background. To maximize the signal-to-noise ratio, you need to reduce background staining due to endogenous enzymes or biotin. Switching from peroxidase to alkaline phosphatase-based detection could be an option. If endogenous biotin proves challenging to block, try non-biotin polymer detection systems, such as ImmPRESS^® Polymer Detection kits. Polymer-based detection offers greater signal-to-noise and superior access to epitopes.

Duration – consider the number of steps involved and the timings around your work schedule. A 30-min incubation time allows for the much-needed beverage break whereas overnight incubations could help your sleep routine (unless you’re a night owl)!

Complexity – how many targets do you plan on staining on the same section? Multiplexed IHC is a different beast and the substrates you choose must be able to distinguish the different antigens. To help you on your journey, we’ve got an IHC Multiplexing Guide and a blog post on getting started with IHC multiplexing if you’re interested! We also have a page dedicated to combining enzyme substrates in our Immunohistochemistry Guide (p.31).

Species cross-reactivity – consider not only the species of primary antibody but also the species of the tissue under investigation. If they are closely related (e.g., rat and mouse), the biotinylated secondary antibody may cross-react with the endogenous IgG leading to background noise. You can overcome this problem using one of the following options:

» Directly label the primary antibody with biotin and detect using the VECTASTAIN^® Elite ABC kit (PK-6100) followed by a horseradish peroxidase (HRP) substrate
» Use a biotinylated secondary antibody specifically adsorbed to remove cross-reacting antibodies of closely related species (e.g., biotinylated anti-mouse IgG, rat adsorbed).
» Use the M.O.M^® (Mouse on Mouse) immunodetection system for applications using mouse primary antibodies on mouse tissue.

Cost – more advanced assay techniques may be less time consuming but can also be more costly. Consider consumables, equipment, and labor costs. There may be times when you need to do something more complex because your experimental considerations require it. For example, if your target is lowly expressed or if you expect endogenous biotin to be an issue, you might want to invest in a more sensitive detection system using a biotin-free system. Although the cost per assay could be higher for these detection systems, rest easy knowing that you’re getting the sensitivity and contrast you need. Think about all the time you’ll be saving and all the pretty pictures you’ll get!
Simplicity – easy-to-use kits with simple straightforward instructions can reduce hands-on time and experimental errors. On the other hand, there’s nothing quite like buying high quality reagents and consumables, setting up your workflow before going home, and coming in the next day already organized and ready to go.

Reproducibility – high quality reagents and methods are required to produce consistent and reliable results. Once you decide on a system that works, stick to it as much as possible.

In Summary

Whew – that was a lot! Take a breath, consider all the points above, and consult the following table to help you choose your detection system:

There are many choices to fit a range of budgets, sensitivities, and performance levels. As pioneers of IHC technology, Vector Laboratories is here to help you choose the correct IHC detection reagents to set you on the path to success and give you confidence in your results.