Kinase signaling networks among cells play crucial roles in many pathophysiological processes, such as tumorigenesis and inflammation. The temporal patterns of kinase signaling, such as oscillations, transient or sustained activity, have been found to carry information that controls cellular responses. To understand the kinase signaling dynamics, single-cell protein analysis can provide a comprehensive perspective of the cellular signaling network. However, with conventional immunofluorescence, the number of protein detection is limited due to the crosstalk between different fluorophores. Herein, we present an iterative immunofluorescence method that utilizes a photocleavable (PC) linker that connects the antibody and fluorophore. After imaging and data acquisition, the PC linker can be cleaved with UV light under a mild condition in the buffer solution. The fluorophore is separated from the antibody and washed away, which allows a new cycle of immunostaining. This approach can be readily combined with the kinase dynamics detection methods. For a proof of concept, we use a pair of chemical probes to continuously monitor the signaling activities of AKT kinase under various perturbations from living cells. Then we apply the iterative immunofluorescence to quantify eighteen proteins along the AKT pathway, that helps us to interpret the AKT signaling dynamics.

Interpretation of cellular kinase signaling dynamics based on multiplexed iterative immunofluorescence

ACS Spring 2022

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