This supplementary material provides detailed experimental data and controls to support the findings in the main article, which discusses the extension of cerebrospinal fluid (CSF) flow to peripheral nerves and its implications for unifying the nervous system. Key points include: 1. **Nanoprobe Injection and Deposition:** - Low volume, high-concentration nanogold injections into the lateral cerebral ventricle yield similar deposition patterns as standard injections. - Nanoprobe distribution in the central nervous system (CNS) recapitulates known CSF flow routes, validating the tracing system. 2. **Control Experiments:** - 1x PBS infusions serve as controls to ensure that staining observed in tissues is not due to the injection itself. - Enhanced histological sections of uninfused tissues show no staining, confirming the specificity of the nanoprobe. 3. **Time and Concentration Effects:** - The effect of nanoprobe concentration on distribution in the peripheral nervous system (PNS) is assessed, showing that higher concentrations lead to more extensive staining over time. - Staining intensity in the PNS increases over time, with the highest levels observed at 4 hours post-infusion. 4. **Subcellular Localization:** - Nanoprobe accumulation is observed in both extracellular and intracellular structures, particularly in collagen fibers and vesicles within the perineurium and endoneurium. - Unmyelinated Schwann cells and macrophages in the PNS exhibit low levels of intracellular nanoprobe staining. 5. **Electron Microscopy:** - Electron microscopy confirms the presence of nanoprobe in extracellular and intracellular vesicles, as well as in the cytoplasm of Schwann cells and axons. - Uninjected nerves show no background staining, validating the specificity of the nanoprobe. These findings collectively demonstrate that CSF flow extends to peripheral nerves, enhancing our understanding of nervous system integration and providing a robust method for tracing and studying nerve tissue.This supplementary material provides detailed experimental data and controls to support the findings in the main article, which discusses the extension of cerebrospinal fluid (CSF) flow to peripheral nerves and its implications for unifying the nervous system. Key points include: 1. **Nanoprobe Injection and Deposition:** - Low volume, high-concentration nanogold injections into the lateral cerebral ventricle yield similar deposition patterns as standard injections. - Nanoprobe distribution in the central nervous system (CNS) recapitulates known CSF flow routes, validating the tracing system. 2. **Control Experiments:** - 1x PBS infusions serve as controls to ensure that staining observed in tissues is not due to the injection itself. - Enhanced histological sections of uninfused tissues show no staining, confirming the specificity of the nanoprobe. 3. **Time and Concentration Effects:** - The effect of nanoprobe concentration on distribution in the peripheral nervous system (PNS) is assessed, showing that higher concentrations lead to more extensive staining over time. - Staining intensity in the PNS increases over time, with the highest levels observed at 4 hours post-infusion. 4. **Subcellular Localization:** - Nanoprobe accumulation is observed in both extracellular and intracellular structures, particularly in collagen fibers and vesicles within the perineurium and endoneurium. - Unmyelinated Schwann cells and macrophages in the PNS exhibit low levels of intracellular nanoprobe staining. 5. **Electron Microscopy:** - Electron microscopy confirms the presence of nanoprobe in extracellular and intracellular vesicles, as well as in the cytoplasm of Schwann cells and axons. - Uninjected nerves show no background staining, validating the specificity of the nanoprobe. These findings collectively demonstrate that CSF flow extends to peripheral nerves, enhancing our understanding of nervous system integration and providing a robust method for tracing and studying nerve tissue.