ST-FFPE-mIF: integrating spatial transcriptomics and multiplex immunofluorescence in formalin-fixed paraffin-embedded tissues using Stereo-seq.

While Stereo-seq-FFPE captures spatial gene expression in formalin-fixed paraffin-embedded (FFPE) samples, it is limited to transcriptomic data. Here, we introduce ST-FFPE-mIF, a protocol that integrates eight-plex immunofluorescence staining with spatial transcriptomics in the same FFPE tissue section. Our approach extends Stereo-seq-FFPE to phenotypic analysis by enabling single-cell resolution through nuclear or membrane-based cell segmentation. We demonstrate how ST-FFPE-mIF supports robust spatial multi-omic profiling in cancer samples, offering a comprehensive view of molecular hierarchies from transcriptome to phenome.