Protocol for observing tunneling nanotube formation and function in both fixed and live primary mouse neurons and microglia coculture system

Microglia and neurons can connect via tunneling nanotubes (TNTs), facilitating the transfer of organelles, vesicles, and proteins. Here, we present a protocol for visualizing murine TNT formation and material transfer between neurons and microglia in both fixed samples and samples for live-cell imaging, as well as for flow cytometry. We describe steps for identifying and measuring TNTs and quantifying the transport of aggregated proteins, such as α-synuclein or tau, between these cells. For complete details on the use and execution of this protocol, please refer to Scheiblich et al.¹.