Standardized Immunofluorescence Imaging of Tunneling Nanotubes and Mitochondrial Transfer Between Astrocytes and...

Mitochondria are essential organelles that regulate energy production, cellular signaling, and metabolic homeostasis in neural cells. Tunneling nanotubes (TNTs) are thin membranous structures that mediate long-distance intercellular communication and facilitate the transfer of cellular components, including mitochondria, between connected cells. Reliable visualization of TNTs and mitochondrial transfer requires careful sample handling because these structures are highly fragile and sensitive to fixation, washing, and imaging conditions. This protocol describes standardized procedures for the fixation, staining, and confocal imaging of TNTs in astrocytes and astrocyte-neuron coculture systems. The workflow includes membrane and cytoskeletal staining for TNT visualization, mitochondrial labeling for tracking mitochondrial localization, and immunofluorescence staining for Miro1 colocalization analysis. Critical steps for preserving TNT morphology, including gentle washing and light-protected handling, are emphasized throughout the procedure. The protocol also outlines imaging approaches for the characterization of TNTs and mitochondria in fixed-cell preparations. These methods provide a reproducible experimental framework for studying TNT formation and mitochondrial transfer between neural cells in vitro.