While highly informative, multi-color labeling presents several challenges:
- Spectral Overlap: Overlapping emission spectra can complicate the interpretation of results. This can be mitigated by using a combination of fluorophores with distinct spectral properties. - Non-Specific Binding: Non-specific antibody binding can lead to background staining, which compromises the clarity of the results. Proper blocking and stringent washing steps are essential to minimize this issue. - Signal Intensity: Different targets may have varying levels of expression, resulting in uneven signal intensities. This can be addressed by optimizing antibody concentrations and using amplification techniques.
