Optimisation of Xenium automated in situ sequencing for PAXgene-fixed tissue samples

Spatial transcriptomics has transformed the study of gene expression in tissue samples, yet its application to non- standard sample preservation formats beyond fresh frozen and FFPE remains underexplored. PAXgene fixation offers advantages for genomic studies by preserving DNA and RNA without crosslinking but poses challenges for high-quality spatial transcriptomics. Here, we present an optimised workflow for applying Xenium, an automated in situ sequencing platform, to PAXgene-fixed paraffin-embedded (PFPE) tissues. Using our newly developed Xenium Tissue Optimisation (XTO) protocol, we systematically tested permeabilisation conditions across multiple mouse and human tissues. We show that pepsin digestion effectively enhances RNA accessibility in PFPE samples, with tuneable digestion times for tissue-specific optimisation, though a compromise may need to be reached between transcript detection rates and maintaining tissue integrity and morphological staining. Our results indicate that, under optimal conditions, PFPE samples can yield comparable or superior spatial RNA transcript detection to formalin-fixed paraffin-embedded (FFPE) samples. This study provides a framework for adapting in situ sequencing to PFPE tissues, broadening the applicability of spatial transcriptomics to archival and prospective sample collections.