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.