Preserving RNA integrity and morphology in whole rat allograft hindlimb sections: an optimized protocol for spatial transcriptomics and immunofluorescence
- 1Department of Plastic and Hand Surgery, Medical Center and Faculty of Medicine, University of Freiburg , ,
- 2Department of Neurosurgery, Medical Center and Faculty of Medicine, University of Freiburg , ,
- 3Translational Neurosurgery, Friedrich-Alexander University Erlangen Nuremberg , ,
Abstract
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.
Related
- Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Tumour, Stroma, and Immune Structures in Human FFPE Tissue
- Multiplex Immunofluorescence-Based Investigation of the Interventional Effect of Bisdemethoxycurcumin on Glial Cell Polarization in Radiation-Induced Brain Injury
- CO061 | IMMUNOFLUORESCENCE MICROSCOPY REVEALS PLATELET- AND RED BLOOD CELL ALTERATIONS AS MORPHOLOGICAL BIOMARKERS IN MYELOPROLIFERATIVE NEOPLASMS
- Association of eplet mismatch load and allograft injury in kidney transplant recipients
- “The box” allograft augmentation for at risk rotator cuff repairs
BibTeX
@article{dreck2026preserving,
title = {{Preserving RNA integrity and morphology in whole rat allograft hindlimb sections: an optimized protocol for spatial transcriptomics and immunofluorescence}},
author = {Marie Dreck and Elena Grabis and Laura Schneider and Maike Lind and Philipp Heinrichs and Thierry Christmann and Nicolas Neidert and Teresa Z. Brose and Sheena Kreuzaler and Franziska Pankratz and Dieter H. Heiland and Steffen U. Eisenhardt and Julia Thomé},
journal = {Frontiers in molecular biosciences},
year = {2026},
month = oct,
doi = {10.3389/fmolb.2026.1910122},
url = {https://europepmc.org/article/PMC/PMC13725282},
note = {CC BY},
}
RIS
TY - JOUR
TI - Preserving RNA integrity and morphology in whole rat allograft hindlimb sections: an optimized protocol for spatial transcriptomics and immunofluorescence
AU - Marie Dreck
AU - Elena Grabis
AU - Laura Schneider
AU - Maike Lind
AU - Philipp Heinrichs
AU - Thierry Christmann
AU - Nicolas Neidert
AU - Teresa Z. Brose
AU - Sheena Kreuzaler
AU - Franziska Pankratz
AU - Dieter H. Heiland
AU - Steffen U. Eisenhardt
AU - Julia Thomé
PY - 2026
DA - 2026/10/19
JO - Frontiers in molecular biosciences
DO - 10.3389/fmolb.2026.1910122
UR - https://europepmc.org/article/PMC/PMC13725282
AB - 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.
SN - 2296-889X
ER -