Michel, H. A.; McCallum, P.; Wu, W.; Lee, J. L.; Luo, S.; Chan, C. N.; Schaffenrath, J.; Yeo, Y. Y.; Yiu, S. P. T.; Wang, Y.; Parmelee, L.; Wang, H.; Burgess, M.; El Ahmar, N.; Zhang, Z. X.; Keane, C.; Lim, T.; Signoretti, S.; Del Rincon, S. V.; Zhao, B.; McIlwain, D. R.; Bai, Y.; Chen, F.; Jiang, S.

Spatial proteomics techniques have revolutionized our understanding of tissue architecture, but are frequently limited by detection sensitivity, bioconjugation limitations, multiplexing capacity, and multi-modal integration. Here we present Protein and nucleic Acid Serial Tyramine Amplification (PASTA), a novel signal amplification approach that significantly enhances detection sensitivity while maintaining compatibility with diverse spatial profiling methodologies. PASTA utilizes horseradish peroxidase (HRP) recruitment pathways to generate tyramine radicals that deposit oligonucleotides, enabling adaptable signal amplification across multiple biomarkers at high-plex via cyclical imaging using complementary fluorophore-labeled oligonucleotides. We demonstrate that PASTA achieves up to 100-fold signal enhancement for markers with minimal background in blank controls. The method is compatible with in situ hybridization for DNA/RNA detection, proximity ligation assays for protein-protein interactions, sequential antibody staining protocols, or any modular combination thereof. PASTA enables antibody rescue of markers with suboptimal signal-to-noise ratios and is versatile in its applications to unconjugated antibodies, and multi-round probe-based RNA detection systems beyond current capabilities. This technique addresses key limitations in spatial-omics by enhancing sensitivity for challenging targets while maintaining compatibility with established multiplexing strategies, providing a versatile, cost-efficient, and valuable tool for comprehensive spatial tissue analysis in both research and clinical applications.