Copeland K.Permata Sari A.I.Copeland J.Jinawath N.Shiao M.S.Mahidol University2025-06-292025-06-292025-09-19STAR Protocols Vol.6 No.3 (2025)https://repository.li.mahidol.ac.th/handle/123456789/110941Here, we present a protocol for quantifying the expression of genes detected by fluorescence in situ 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.¹NeuroscienceBiochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyArticleSCOPUS10.1016/j.xpro.2025.1039142-s2.0-10500852770526661667