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

• Published in: STAR protocols Vol. 4; no. 4; p. 102744
• Main Authors: Haan, Kirk D., Fisher, Thomas E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-12-2023; Elsevier
• Subjects: Cell culture; Cell isolation; Cell Membrane; Immunohistochemistry; Ion Channels; Membrane Proteins; Microscopy; Neurons; Neuroscience; Protocol; Cell culture; Neuroscience; Microscopy; Cell isolation
• 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
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•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.