Powell Emma L, Salecker Iris
Visual Circuit Assembly Laboratory, The Francis Crick Institute, London, UK.
Methods Mol Biol. 2020;2047:137-152. doi: 10.1007/978-1-4939-9732-9_8.
Visualization of single neurons and glia, as well as neural lineages within their complex environment is a pivotal step towards uncovering the mechanisms that control neural circuit development and function. This chapter provides detailed technical information on how to use Drosophila variants of the mouse Brainbow-2 system, called Flybow, for stochastic labeling of individual cells or lineages with different fluorescent proteins in one sample. We describe the genetic strategies and the heat shock regime required for induction of recombination events. Furthermore, we explain how Flybow and the mosaic analysis with a repressible cell marker (MARCM) approach can be combined to generate wild-type or homozygous mutant clones that are positively labeled in multiple colors. This is followed by a detailed protocol as to how to prepare samples for imaging. Finally, we provide specifications to facilitate multichannel image acquisition using confocal microscopy.
在其复杂环境中可视化单个神经元、神经胶质细胞以及神经谱系，是揭示控制神经回路发育和功能机制的关键一步。本章提供了详细的技术信息，介绍如何使用小鼠Brainbow-2系统的果蝇变体Flybow，在一个样本中用不同荧光蛋白对单个细胞或谱系进行随机标记。我们描述了诱导重组事件所需的遗传策略和热休克方案。此外，我们解释了如何将Flybow与可抑制细胞标记的镶嵌分析（MARCM）方法相结合，以生成用多种颜色进行阳性标记的野生型或纯合突变克隆。接下来是关于如何制备成像样本的详细方案。最后，我们提供了使用共聚焦显微镜进行多通道图像采集的规格说明。
