Nagendran Tharkika, Poole Valerie, Harris Joseph, Taylor Anne Marion
UNC Neuroscience Center; UNC/NC State Joint Department of Biomedical Engineering, UNC.
Xona Microfluidics, LLC.
J Vis Exp. 2018 Nov 2(141). doi: 10.3791/58421.
Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for >3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.
微制造方法将神经元分隔开来，已成为许多神经科学家的重要工具。本方案描述了使用市售预组装塑料芯片对培养的原代大鼠海马神经元进行分隔的方法。这些塑料芯片尺寸与标准显微镜载玻片相同，适用于高分辨率、实时和荧光成像。本方案展示了如何使用编码荧光蛋白的改良狂犬病病毒通过分离的轴突对神经元进行逆行标记，在一个隔室内创建隔离的微环境，以及在芯片上进行轴突切断和免疫细胞化学操作。神经元在塑料芯片内培养超过3周，表明这些芯片适用于长期神经元培养。
