Despite its advantages, multiplex IHC also presents several challenges: - Antibody Cross-Reactivity: Ensuring that antibodies do not cross-react with non-target antigens is crucial for accurate interpretation of results. - Signal Overlap: Fluorescent signals from different markers can overlap, making it difficult to distinguish between them. Proper selection of fluorophores and optimization of imaging settings are essential. - Complex Protocols: The sequential staining and detection process can be time-consuming and requires meticulous optimization to prevent interference between different steps.
