Multiplex IHC Optimization: Antibody Titration & Fluorophore Pairing | CST Tech Tips

When designing an antibody panel for multiplex IHC, it’s important to optimize each antibody’s dilution and which fluorophore it’s paired with. This will help your staining achieve a balanced set of signals.
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Transcript:
How do I determine the optimal antibody concentration, and how can I get a balanced set of signals in fluorescent multiplex IHC? I'm Jen, senior research associate at Cell Signaling Technology, and this is CST Tech Tips. Before your first fluorescent multiplex IHC experiment, you'll need to plan your protocol and optimize parameters such as antibody dilution and the pairing of fluorophores for each target.
If you're just starting out with the multiplex IHC protocol, you may want to check out our first video for an overview of protocol optimization. • 5 Tips to Start Your M... Otherwise, keep watching here for more details about antibody titration and order optimization. And take a second to like this video and subscribe for more Tech Tips on IHC and other techniques.
Any time you bring an IHC validated antibody from a single stain protocol to a multiplex protocol with tyramide amplification, a major consideration is the relative strength of staining of each antibody and fluorophore in your panel. The optimal dilution for each antibody may differ between chromogenic and fluorescent detection protocols, thanks to the higher signal amplification provided by tyramide-fluorophore conjugates. We recommend performing titrations for each primary antibody in your panel, using a moderately intense tyramide fluorophore conjugate in a single plex setting.
Once you've determined the optimal dilution for each antibody, your next goal is to obtain a balanced set of signals. Some of the targets in your panel may be more abundant than others, and the brightness of the fluorophores in your panel can also vary. Ideally, you would want to pair the antibodies with the strongest labeling or most abundant expression of the target protein with the weakest available tyramide-fluorophore conjugates. Conversely, you can pair the weakest-staining antibodies with the brightest conjugates.
We recommend setting up a matrix composed of each primary antibody applied at its optimized dilution, paired with each available fluorophore. Pick antibody fluorophore pairs for each target that exhibit strong signal intensity and robust signal to noise ratio. Then, once you assemble your panel and start multiplexing, it's a good practice to compare single plex fluorescent IHC for each antibody to the multiplex panel, to confirm that each antibody is performing as expected in the multiplex protocol.
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