SignalStar™ Multiplex IHC Antibody & Fluorophore Pairing Considerations

The SignalStar Multiplex IHC Panel Builder provides automated panel design, pairing antibodies to fluorophores and imaging rounds with efficient accuracy. Panel builder results can be edited for complete control of design. If you choose to modify your panel, keep these considerations in mind:

Protein Abundance: Channels 594 and 647 are brightest, and should be paired with antibodies binding the least abundant antigens. Channel 750 has a lower signal intensity, and the panel builder will prohibit some antibodies from pairing with the 750 channel.

Autofluorescence: Antibodies expected to detect low abundance proteins in your tissue should be in channels less affected by autofluorescence. Channel 488 is most impacted by autofluorescence and should be avoided for these biomarkers.

Imaging Rounds: While the majority of antibodies should be strong and clean regardless of imaging round, signal intensity may be impacted by low target expression levels and poor tissue quality. For best results, antibodies against lower abundance proteins should be placed in the first imaging round, while antibodies against higher abundance markers should be placed in the second imaging round. The panel builder may prohibit some antibodies from being used in certain imaging rounds.

Imaging Instrumentation: To avoid spectral bleed-through, consider the spectral characteristics of your fluorescence microscope. Epifluorescent, confocal, and scanning fluorescence microscopes can typically distinguish between the SignalStar channels:

If your final panel includes all four (4) fluorescent channels, in addition to DAPI, then you'll need to confirm your fluorescent imaging instrumentation can specifically detect and separate these channels based on these parameters.

Spectral Unmixing: The antibodies, reagents, and protocols included in your SignalStar Multiplex IHC Panel are optimized to minimize spectral overlap. To further ensure that fluorescence from individual antibodies can be differentiated, we recommend creating a spectral library using slides stained individually with the channels described above.

If the characteristics of your imaging instrument are not ideal for separating these fluorescent channels, a spectral library can be used to computationally unmix fluorescent signals.