Protocol to visualize ion channel trafficking in acutely isolated rodent neurons using live-cell immunocytochemistry

• Published in: STAR protocols 2023-12, Vol.4 (4), p.102744-102744, Article 102744
• Main Authors: Haan, Kirk D., Fisher, Thomas E.
• Format: Article
• Language: English
• Subjects: Cell culture; Cell isolation; Cell Membrane; Immunohistochemistry; Ion Channels; Membrane Proteins; Microscopy; Neurons; Neuroscience; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102744

Here, we present a protocol for live-cell immunocytochemistry to demonstrate reversible translocation of ion channels to the neuronal cell surface. We describe steps for cell preparation and isolation, experimental treatment, antibody binding prior to fixation, specific pipetting techniques, troubleshooting, and expected outcomes of correct use of the protocol. This protocol will be useful to study regulated translocation of ion channels and other membrane proteins. For complete details on the use and execution of this protocol, please refer to Haan et al.1 [Display omitted] •Steps described for tracking ion channel movement in acutely isolated neurons•Imaging of channel internalization via external epitope antibodies•Visualization of regulated channel trafficking in neurons•Visualization of regulated channel trafficking in cultured cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for live-cell immunocytochemistry to demonstrate reversible translocation of ion channels to the neuronal cell surface. We describe steps for cell preparation and isolation, experimental treatment, antibody binding prior to fixation, specific pipetting techniques, troubleshooting, and expected outcomes of correct use of the protocol. This protocol will be useful to study regulated translocation of ion channels and other membrane proteins.