FFPE Tissue Processing for Multimodal Imaging Assays (Phenocycler-Fusion + H&E) following Xenium In Situ Gene Expression v1

Xenium In Situ Gene Expression assay from 10X Genomics enables multiplexed imaging of RNA transcripts in situ. There are several antibody based multiplexed imaging technologies that enable multiplexed protein detection. Hematoxylin and Eosin (H&E) staining is a gold standard method for observing tissue morphology. In order to generate a highly multimodal imaging dataset, we developed a protocol to prepare tissue slides for multiplexed imaging on the Phenocycler-Fusion platform and H&E staining following a Xenium run. Although the Xenium protocol includes a decrosslinking step which is akin to antigen retrieval, a second round of antigen retrieval, as outlined in this protocol, is necessary for epitope unmasking and accurate protein detection by antibody based methods. The steps for slide storage and tissue processing for multiplexed imaging can also be applied to imaging assays on other platforms.