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SignalStar Multiplex IHC Frequently Asked Questions

Questions

Answers

How are the SignalStar Multiplex IHC Kits & Reagents validated?

CST thoroughly validates each antibody available in the SignalStar Multiplex IHC Panel Builder menu. Various combinations of antibodies are tested through titration and fluorophore pairing, and in both rounds of imaging. Testing is performed on a variety of tumors and tissue types. We also rigorously test the parent antibodies used in the traditional chromogenic assay, as they serve as the foundation of this fluorescent assay.

Does this assay work on frozen tissue?

SignalStar Multiplex IHC Kits & Reagents haven’t yet been validated for use in frozen tissues. We’re in the process of validating our antibodies and protocols for use in fresh or frozen tissue.

Do you have anti-mouse antibodies available?

SignalStar Multiplex IHC Kits & Reagents haven’t yet been validated for use in mouse tissues. We’re in the process of validating mouse-reactive antibodies.

I don’t see my target of interest in your menu of available antibodies. Can I still use it in my panel in some way?

SignalStar Multiplex IHC Kits & Reagents haven’t yet been validated for use with antibodies outside of our menu. We’re in the process of developing custom solutions for using your own antibodies in the SignalStar Multiplex IHC assay.

Can I combine antibodies used in this assay with direct conjugates?

SignalStar Multiplex IHC Kits & Reagents haven’t been validated for use in combination with direct conjugates. It’s likely possible to incorporate direct conjugates into your protocol. However, because the SignalStar reagents benefit from fluorescent signal amplification, there may be spectral bleed-through that results from using the assay with conjugates that are not amplified.

When comparing my SignalStar staining to the chromogenic staining on a serial section, I see more positive cells. How do I know if this excess staining is correct?

During the course of optimization, we've found that fluorescent staining may show higher %-positivity than chromogenic staining. To ensure any excess staining is specific, confirm that the correct subcellular localization and co-localization with other stains are demonstrated. For example, if all CD8+ cells are CD3+, any excess CD8+ staining compared to the chromogenic is most likely correct.

How long after the completion of staining can I wait to image my slides?

For Imaging Round 1, the staining should show robust signal when imaged up to 8 hr post completion of staining. For Imaging Round 2, imaging should be performed as close to the completion of staining as possible, but should remain robust for up to 8 hr.

Do I need to optimize the SignalStar Kits & Reagents for the type of tissue that I'm using?

The SignalStar Multiplex IHC Kits & Reagents have been optimized with respect to fluorophore pairing and order of antibodies. As tissues vary in quality and expression level of targets, increasing the concentration of antibodies in your panel by 2-fold or decreasing by 0.5 fold can help achieve optimal signal in your experiments.

What is an appropriate positive control to include in this assay? Are multiple controls necessary?

Any tissue shown to be positive for each target via chromogenic IHC can serve as a positive control tissue. Each target will therefore require a positive control, which may sometimes necessitate multiple controls. For optimal comparison, the sections should be as close to serial as possible.

For any additional questions, please contact our SignalStar group directly at support@cellsignal.com.