Robust carotid artery perfusion protocol for turtle brain fixation enabling multiscale imaging from magnetic resonance to super-resolution microscopy

• Stepwise protocol for freshwater turtle brain fixation and dissection. • Perfusion via common carotid arteries, overcoming limitations of transacrdial approch. • Resulting in rapid and homogeneous brain fixation. • Validated across imaging modalities, from whole-brain MRI to super-resolution STED imaging. • Demonstrates first label-free stimulated Raman histology (SRH) in turtle brain tissue. Reptiles, particularly turtles, are increasingly recognized as valuable models in comparative and evolutionary neuroscience; however, standardized methods for brain perfusion and fixation remain lacking. Here, we present the first detailed, stepwise protocol for turtle brain perfusion via cannulation of the common carotid arteries after decapitation. This approach overcomes the anatomical and technical limitations of transcardial perfusion and enables rapid and uniform delivery of fixatives directly to the cerebrovascular system. The protocol consistently yields pale, blood-free brains with well-preserved gross morphology. Uniform parenchymal fixation was validated using high-contrast magnetic resonance imaging (MRI), consistent immunohistochemical labeling, label-free stimulated Raman histology (SRH), and preservation of fine neuronal structures revealed by super-resolution stimulated emission depletion (STED) microscopy. In addition, we describe a comprehensive procedure for brain dissection from the skull. Together, these methods provide a robust framework for homogeneous brain fixation compatible with advanced multiscale imaging.