1. Multiplex Protein Imaging through PACIFIC: Photoactive Immunofluorescence with Iterative Cleavage
- Author
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Ji, Fei, Hur, Moises, Hur, Sungwon, Wang, Siwen, Sarkar, Priyanka, Shao, Shiqun, Aispuro, Desiree, Cong, Xu, Hu, Yanhao, Li, Zhonghan, and Xue, Min
- Subjects
Generic health relevance - Abstract
Multiplex protein imaging technologies enable deep phenotyping and provide rich spatial information about biological samples. Existing methods have shown great success but also harbored trade-offs between various pros and cons, underscoring the persisting necessity to expand the imaging toolkits. Here we present PACIFIC: photoactive immunofluorescence with iterative cleavage, a new modality of multiplex protein imaging methods. PACIFIC achieves iterative multiplexing by implementing photocleavable fluorophores for antibody labeling with one-step spin-column purification. PACIFIC requires no specialized instrument, no DNA encoding, or chemical treatments. We demonstrate that PACIFIC can resolve cellular heterogeneity in both formalin-fixed paraffin-embedded (FFPE) samples and fixed cells. To further highlight how PACIFIC assists discovery, we integrate PACIFIC with live-cell tracking and identify phosphor-p70S6K as a critical driver that governs U87 cell mobility. Considering the cost, flexibility, and compatibility, we foresee that PACIFIC can confer deep phenotyping capabilities to anyone with access to traditional immunofluorescence platforms.
- Published
- 2023