From Stains to Spectra: Multiplexed Immunohistochemistry and Mass Spectrometry Imaging for Spatial Proteomics in Biomedicine.

Advances in spatial proteomics have fundamentally transformed the analysis of complex tissues by enabling the simultaneous interrogation of protein expression and spatial organization within intact biological samples. Early immunohistochemical approaches were limited by low multiplexing capacity, restricting the ability to capture cellular heterogeneity and microenvironmental interactions. Recent technological developments have markedly expanded multiplexing power, allowing the parallel detection of dozens of protein markers while preserving tissue architecture and spatial context. Multiplexed immunohistochemistry and immunofluorescence techniques have substantially increased the number of detectable targets in a single tissue section and enabled high-resolution mapping of cellular phenotypes. In parallel, mass cytometry-based imaging and mass spectrometry imaging platforms have introduced label-free or metal-tagged strategies that overcome spectral overlap, further extending multiplexing capabilities and quantitative robustness. These approaches enable comprehensive spatial profiling of tumor ecosystems and tissue architecture in both normal and diseased states. The integration of immunohistochemistry with mass spectrometry-based spatial proteomics has led to hybrid workflows that combine targeted molecular specificity with unbiased proteome-wide analysis. This convergence has expanded the analytical depth of spatial biology, supporting applications ranging from biomarker discovery and drug distribution studies to mechanistic insights into cancer progression, immune regulation, and neurodegenerative disease. Collectively, the continuous increase in multiplexing capacity across imaging and mass spectrometry platforms has positioned spatial proteomics as a powerful and versatile framework for biomedical research, with growing translational and clinical relevance.