Protocol for simultaneous detection of mRNAs and (phospho-)proteins with ARTseq-FISH in mouse embryonic stem cells

Understanding the molecular signatures of individual cells within complex biological systems is crucial for deciphering cellular heterogeneity and uncovering regulatory mechanisms. Here, we present a protocol for simultaneous multiplexed detection of selected mRNAs and (phospho-)proteins in mouse embryonic stem cells using spatial single-cell profiling. We describe steps for employing single-stranded DNA (ssDNA)-labeled antibo’dies, padlock probes, and rolling circle amplification to achieve simultaneous visualization of mRNAs and (phospho-)proteins at subcellular resolution. This protocol has potential application in identifying cells in heterogeneous biological microenvironments.