A comprehensive illustrated protocol for clearing, mounting, and imaging leaf venation networks

Premise: Leaf venation network architecture can provide insights into plant evolution, ecology, and physiology. Venation networks are typically assessed through histological methods, but existing protocols provide limited guidance on processing large or challenging leaves. Methods and results: We present an illustrated protocol for visualizing whole leaf venation networks, including sample preparation, clearing, staining, mounting, imaging, and archiving steps. The protocol also includes supply lists, troubleshooting procedures, safety considerations, and examples of successful and unsuccessful outcomes. The protocol is suitable for a wide range of leaf sizes and morphologies and has been used with all major plant groups. Conclusion: We provide a workflow for obtaining high-quality mounts and images of venation networks of a wide range of species, using readily available materials.