A Hitchhiker's guide to high-dimensional tissue imaging with multiplexed ion beam imaging.

Advancements in multiplexed tissue imaging technologies are vital in shaping our understanding of tissue microenvironmental influences in disease contexts. These technologies now allow us to relate the phenotype of individual cells to their higher-order roles in tissue organization and function. Multiplexed Ion Beam Imaging (MIBI) is one of such technologies, which uses metal isotope-labeled antibodies and secondary ion mass spectrometry (SIMS) to image more than 40 protein markers simultaneously within a single tissue section. Here, we describe an optimized MIBI workflow for high-plex analysis of Formalin-Fixed Paraffin-Embedded (FFPE) tissues following antigen retrieval, metal isotope-conjugated antibody staining, imaging using the MIBI instrument, and subsequent data processing and analysis. While this workflow is focused on imaging human FFPE samples using the MIBI, this workflow can be easily extended to model systems, biological questions, and multiplexed imaging modalities.

Combined protein and nucleic acid staining in tissues with PANINI.

We present here a detailed protocol for PANINI (protein and nucleic acid in situ imaging), a technique that enables the concurrent staining of protein and nucleic acids in archival tissue sections. PANINI utilizes an optimized antigen retrieval strategy that forgoes protease treatment while retaining high sensitivity of nucleic acid detection down to single genomic events. While the protocol here is geared toward standard fluorescent microscopes with 3-4 available channels, PANINI is compatible with many commercial multiplexed tissue imaging modalities. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2022).