Protocol for integrating immunohistochemistry and H&E annotations with Xenium data at single-cell resolution.

Targeted investigation of spatial transcriptomic data benefits from versatile integration of customized external information. Here, we present a protocol for embedding irregular field-of-view annotations and immunohistochemistry data into a Xenium dataset at the single-cell resolution using open-source tools. We detail steps for image registration and demonstrate how to integrate spatially aligned information into a Scanpy-compatible anndata object using a head and neck squamous cell carcinoma sample. This protocol facilitates targeted analysis of spatial transcriptomic data.