Despite its advantages, multiplex staining presents several challenges:
1. Antibody Cross-Reactivity: Ensuring that antibodies do not cross-react with non-target antigens is crucial for accurate results.
2. Fluorophore Overlap: In immunofluorescence, spectral overlap between fluorophores can complicate the interpretation of results. Careful selection of fluorophores and compensation techniques are required.
3. Tissue Autofluorescence: Endogenous tissue autofluorescence can interfere with signal detection, necessitating the use of quenching agents or specialized imaging techniques.
4. Standardization: Consistency in staining protocols and antibody validation is essential to ensure reproducibility.
