PANINI: Combined protein and nucleic acid imaging in tissues

Sizun Jiang.


Simultaneous detection of DNA, RNA and proteins within the native tissue microenvironment is a trifecta that promises to deliver valuable insights into fundamental aspects of the biological processes underlying health and disease. Recent technical advances in both in situ hybridization and highly multiplexed imaging have enabled this combined analysis, yet many approaches are limited by poor detection of low copy numbers or difficulties owing to protein epitope loss during tissue processing. Our approach, termed PANINI (protein and nucleic acid in situ imaging), enables the detection of low-copy nucleic acids while preserving protein epitopes in intact tissues. This is achieved by bypassing the protease digestion step, which is used in various in situ hybridization assays to increase the accessibility of target nucleic acids by disrupting the packed architecture of tissue matrices and nucleic acid-binding proteins. PANINI starts with a protease-free antigen retrieval protocol before the application of a highly sensitive custom branched-chain amplification method in which horseradish peroxidase catalyses the deposition of tyramine-linked reporter haptens for nucleic acid labelling. These labelled nucleic acid targets, in conjunction with other protein targets, can then be detected using various antibody-based imaging platforms. We demonstrated that PANINI is compatible with multiplex spatial imaging platforms, including conventional microscopes, multiplexed ion beam imaging (MIBI), co-detection by indexing (CODEX) and multispectral imaging on the Vectra Polaris platforms1.