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Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.