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. We aren't able to test all multiplex configurations or tissues. Please visit the Technical Support page to find answers for any additional questions.
SignalStar Multiplex IHC kits and 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.
Yes, please select “Human” in the first step of the SignalStar Multiplex IHC Panel Builder to see our human-reactive menu.
Yes, please select “Mouse” in the first step of the SignalStar Multiplex IHC Panel Builder to see our mouse-reactive menu.
SignalStar Multiplex IHC kits and 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 SignalStar assays.
SignalStar mIHC is a non-destructive technology. Therefore, in order to use your antibodies of interest, you may perform direct immunofluorescence on the same tissue after the SignalStar assay. The SignalStar™ Fluorescence Removal Kit #32722 enables you to remove the fluorescent oligos after SignalStar mIHC in order to stain and visualize direct immunofluorescence.
SignalStar Multiplex IHC kits and reagents have been validated for use in combination with direct conjugates. The SignalStar™ Fluorescence Removal Kit #32722 enables you to remove the fluorescent oligos after SignalStar mIHC in order to stain and visualize direct immunofluorescence. We have found that many direct conjugates against strong cell surface markers work well when used in this manner. Please see our recent poster for more information. In addition, please see our line of directly conjugated antibodies for use in direct immunofluorescence imaging.
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.
For Imaging Round 1, the staining should show robust signal when imaged up to 8 hours 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 hours.
The SignalStar Multiplex IHC kits and reagents have been optimized with respect to fluorophore pairing and order of antibodies. As tissues vary in quality and expression level of biomarkers, 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.
Any tissue shown to be positive for each marker 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 optimal results, we don't recommend deviating from SignalStar protocols. However, if you don't have access to the antigen retrieval method detailed in the protocol, you may try using a microwave, which has shown to result in reduced fluorescent signal. We don't recommend using a microwave when detecting low abundance markers, and can't guarantee your result with this alternative method.
To perform antigen retrieval using a microwave:
While we utilize a wide variety of tumor and tissue types during the course of our optimization process, we can't account for all tissues and expression levels. This assay should work in FFPE tissue 4-5 µM in thickness, assuming that our recommended protocol is followed. Furthermore, due to the high level of amplification that this assay provides, even very high autofluorescence levels may be overcome with the resulting strong, specific signal.
Visit the Technical Support page to search for SignalStar-related troubleshooting information and answers to technical questions.
Cell Signaling Technology, CST, and SignalStar are trademarks of Cell Signaling Technology, Inc. All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
U.S. Patent No. 10,781,477, foreign equivalents, and child patents deriving therefrom.
