Fixation on Histology

IHC is an important tool that histology scientists use to obtain information on protein expressions needed for diagnostics and treatments. With multiplex IHC stains, more than one primary antibody is used on a single slide, so more information can be extracted during one test. In the NSH webinar, Multiplex IHC: The Advantages of Doubling Up, Christine Easson, Applications and Workflow Specialist/Canadian Marketing Coordinator at Agilent Technologies goes into detail about how multiplex IHC works and why it’s worth incorporating into your lab.

Pros and Cons of Multiplex IHC Techniques

Like any other technique that histologists use in the lab, IHC methods each come with their own unique benefits and drawbacks. Easson discussed a few of these techniques to give you a realistic picture of what to expect when using them.

Sequential multiplex. This is where histology scientists build on the existing foundation from the single IHC methods by adding layers. This is done by removing the immune complex from the first reaction and stripping off the complex that was formed with the detection system. 

One of the benefits of this method is that it’s species independent, so it doesn’t matter what your primary antibody is. Whether you’re using a guinea pig, mouse or rabbit, as long as your detection system reacts to it, you can choose which species is best for the tests you’re doing. In addition, sequential multiplex gives you a choice of detection types. 

However, the sequential multiplex technique does take more time than its single counterpart, which can cause its share of challenges.

“It does increase the potential for tissue damage because we’re kind of manhandling that tissue section for a longer period of time, so there’s more rinses. All of this opens the door to a little bit of tissue damage if that section hasn’t been well processed or adequately dried to a sticky slide,” Easson explained. “There’s also the potential for background staining. If the previous immune complex isn’t sufficiently removed during the elution step, you're opening the door for a little bit of background.”

Despite these potential issues, Easson says the sequential multiplex process is still worthwhile.

“None of these things are deal breakers,” she said. “They’re all easily overcome, but they are things that you have to take into account.”

Concurrent multiplex. Also called simultaneous multiplex, the concurrent multiplex technique is when multiple antibodies are applied all at one time. There should be two different antibody species together, such as a mouse and rabbit.

A major benefit of this method is that it takes less time because you’re doing all the layers at once. However, the time it takes depends heavily on the species being used, so combinations of antibodies should be strongly considered when designing a protocol.

Easson emphasizes that stability can also be an issue when using this technique.

“You’re going to be using alkaline phosphatase and the chromatins tend to be a little bit unstable,” Easson said. “You’re going to need to take that into consideration in handling those once the staining procedure is finished.”

Multiplex IHC is an approach worth exploring thanks to its ability to deliver richer data from a single tissue section, saving both time and valuable samples. While each technique has its own considerations, understanding their strengths and limitations can help labs choose the method that best fits their needs. For a deeper look at how multiplex IHC works in practice—and how different laboratories are successfully implementing it—watch the NSH webinar Multiplex IHC: The Advantages of Doubling Up, where Easson also shares a real-world case study showcasing the technique in action.