Protocol for spatial analysis of multiple markers across adjacent tissue sections captured by RNAscope using CellProfiler.

Here, we present a protocol for quantifying the expression of genes detected by fluorescence insitu hybridization at single-cell spatial resolution using an imaging analysis pipeline developed based on CellProfiler software. We describe steps for comparing gene expression between cancer samples with and without metastasis. We detail procedures for detecting target transcripts using RNAscope multiplex fluorescent assays and visualization using Vectra Polaris Automated Quantitative Pathology Imaging. This protocol also provides an alternative option to analyze signal interactions across adjacent tissue sections. For complete details on the use and execution of this protocol, please refer to Sari et al..