Multiplex IHC Optimization: Antibody Order | CST Tech Tips

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Your fluorescent multiplex IHC staining may be affected by the order in which you apply the antibodies in your panel. This video outlines strategies to achieve balanced signals for all your targets.
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Partial transcript:
How does the order of the antibodies in my multiplex IHC panel affect staining results, and how can I optimize my antibody panel? I’m Jen, senior research associate at Cell Signaling Technology, and this is CST Tech Tips.
The protocol we recommend for fluorescent multiplex immunohistochemistry, or mIHC, uses a serial staining strategy, where each antibody is incubated with the sample one at a time, followed by deposition of a tyramide-based fluorophore to enable detection.
If you missed our mIHC overview video, click this pop-up link in the corner to see an animation of how serial labeling works, and then come back to this video.
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In this video, I’ll refer to each primary antibody incubation step as a “slot”, and a collection of antibodies as a “panel”. Your goal in mIHC is to obtain a balanced set of signals from all of your antibodies, while also accurately reporting the biology in your sample. In the course of your research, you may decide to create new combinations of antibodies in your multiplex panel. This can present challenges, because some antibodies perform well in both early and late incubation slots, while others do not.
To illustrate why this may be a problem, let’s consider a multiplex panel with six labels, and look at the epitope for the first label in your panel, and compare it to the last epitope. Before the first labeling slot, the sample is subjected to an antigen retrieval step, by boiling in a microwave for 10 minutes. This helps the antibody to access its epitope in the sample, and it’s also routinely used in single stain IHC.
So, the epitope for your first labeling slot will experience 10 minutes of heat time by the time its antibody incubation step occurs. Then, after the tyramide-fluorophore complexes are deposited, the antibody is stripped, or removed from the sample, again with a microwave heating step. This process is repeated for each antibody, such that epitopes for later labeling slots experience more heating rounds. So, in the sixth labeling slot, the sample has been heated six times, for a total of an hour, before this epitope ever sees its antibody!
The implication of this situation is that an antibody that performs well in chromogenic IHC may have lower than expected signal in multiplex IHC even with tyramide amplification. This is dependent on how sensitive the epitope is to degradation by heating, and how sensitive it is to epitope masking, a phenomenon which I’ll define later in this video.
When receiving a new antibody for use in mIHC, you can evaluate epitope sensitivity by performing a mock protocol with serial tissue sections or cell pellets with known expression.
Each of your test samples will go through all of the heating steps in your protocol, depicted in red in this diagram.
You'll stain samples with a single primary antibody, secondary antibody, and fluorophore, depicted here as blue, green and orange, but apply them in different heating slots for each test.
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