Preserving RNA integrity and morphology in whole rat allograft hindlimb sections: an optimized protocol for spatial transcriptomics and immunofluorescence

Vascularized composite allotransplantation holds great promise in reconstructive surgery, offering the potential to significantly improve the quality of life for patients with extensive facial injuries or limb amputations. However, this type of transplantation remains challenging due to the high complexity of multiple tissue types and the diverse immunological responses within the graft. For investigation of rejection patterns valid experimental models, including rodent hindlimb transplantation, are crucial. Spatial transcriptomics provides a powerful approach to investigate gene expression patterns associated with ongoing rejection in their spatial context. To date, spatial transcriptomics has not yet been described for rat hindlimb transplantation. In this study, we describe an optimized protocol for spatial transcriptomics and immunofluorescence staining of rat hindlimb cross-sections. The rat model enables a detailed investigation of the spatial cellular organization across the entire transplanted limb, encompassing muscle, skin, nerve, and vascular structures. We present a refined workflow encompassing tissue embedding, cryosectioning, immunofluorescence staining, permeabilization timing, and RNA quality control, followed by the workflow of the spatial transcriptomics procedure. This protocol establishes a reproducible and robust framework for the spatial characterization of vascularized composite allografts and provides a foundation for future spatial studies of immune-mediated rejection and musculoskeletal disease in rodent limb models.