Protocol for SUM-PAINT spatial proteomic imaging generating neuronal architecture maps in rat hippocampal neurons

To unravel the complexity of biological processes, it is necessary to resolve the underlying protein organization down to single proteins. Here, we present a protocol for secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a DNA-PAINT-based super-resolution microscopy technique that is capable of resolving virtually unlimited protein species with single-protein resolution. We describe the steps to prepare neuronal cultures, troubleshoot and conduct SUM-PAINT experiments, and analyze the resulting feature-rich neuronal cell atlases using unsupervised machine learning approaches. For complete details on the use and execution of this protocol, please refer to Unterauer et al.¹.