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5 Tips to Start Your Multiplex IHC Optimization | CST Tech Tips

When starting a multiplex IHC experiment, optimizing your protocol is critical to your success. In this Tech Tip, we’ll highlight 5 steps to optimize your protocol. 👉Link to the CST protocol for fluorescent mulitplex IHC: https://cst-science.com/e7obrg 👉Antibody Titration & Fluorophore Pairing video:    • Multiplex IHC Optimization:  Antibody...   👉Get in touch with a CST scientist: https://cellsignal.com/support 👉Subscribe for more Tech Tips: http://youtube.com/user/cellsignaldot... 💡 Get insights and advice on techniques, publishing, and navigating a scientific career: https://www.cellmentor.com/ Transcript: What do I need to know about the differences between fluorescent multiplex IHC, and chromogenic IHC protocols, and how do I optimize my multiplex protocol? Hi, I’m Jen, senior research associate at Cell Signaling Technology, and this is CST Tech Tips. Immunohistochemistry, or IHC for short, uses antibodies to detect and visualize antigens in cells or tissue. Multiplex immunohistochemistry allows you to get more data from paraffin-embedded tissue samples. For the purposes of this video, I’ll use mIHC to refer to fluorescence-based multiplex IHC, which allows you to achieve the highest “plex” and characterize 6 or more targets in one sample, with sensitive detection. In this video, I’ll introduce five key optimization steps that can affect the success of your mIHC experiment. But first, why consider multiplex over single stain IHC? If the direction of your research involves questions about the interactions of different cell types, or if you need to assess expression or colocalization of multiple biomarkers in tissue, mIHC can be leveraged to gain these kinds of insights. We have developed a mIHC protocol using tyramide fluorophore detection, we’ll include a link in the text below this video (https://cst-science.com/e7obrg). Our multiplex IHC uses a serial labelling strategy. Each round of labeling includes steps for primary and secondary antibody incubation, followed by deposition of tyramide fluorophore conjugates.Then, the antibodies are stripped, or removed from the sample with heating in a microwave oven, while the deposited fluorophores remain covalently bound in the sample. Each labeling round incorporates a primary antibody for a different protein of interest paired with a different fluorophore. This strategy offers flexible experimental design, plus the sensitive detection afforded by tyramide. It is critical to choose highly validated antibodies to ensure you get reliable results. Combining antibodies that lack specificity in tissue is sure to yield staining that’s impossible to interpret. Generally, antibodies that have been validated in chromogenic IHC can also be used in multiplex IHC, after you have performed protocol optimization. OK, now we’re ready to introduce the five key protocol optimization steps you should consider when setting up your panel.As with any optimization, you should test one variable at a time. First, titration for each primary antibody needs to be performed in the context of tyramide detection to identify the dilution that yields optimal signal intensity and signal to noise ratio. The optimal dilution may be different for tyramide based detection compared to chromogenic detection. Anytime you will be bringing an antibody into a mutliplex protocol, or designing a new antibody panel, titration is recommended. Second is fluorophore pairing. The goal of this step is to get a balanced set of signals. A good practice is to start by pairing antibodies for targets with low expression with the brightest fluorophores in your panel, while pairing the antibodies against the most abundant targets with weaker fluorophores. Order optimization refers to the order each primary antibody is applied in the panel, which may affect the strength of staining either positively or negatively, depending on the epitope and antibody. Again, we’ll cover this topic in more depth in a future video. Strip testing is used to confirm efficient removal of the primary and secondary antibody. This is important because incomplete stripping can result in fluorophores detecting targets from the current round plus targets from previous rounds, leaving you with misleading results at the end of your experiment. And finally, compare single staining for each antibody performed in parallel with multiplex staining. This serves as a quality control step - if you see significantly reduced signal in the mutliplex compared to single stain, or an increase or decrease in number of cells staining positive, it can be an indication that your protocol needs further optimization. About CST: https://cellsignal.com/about #CSTTechTips, #immunohistochemistry, #antibody #mIHC