Flybow to Dissect Circuit Assembly in the Drosophila Brain: An Update
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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.
KeywordsDrosophila Brainbow Confocal laser scanning microscopy Genetics Immunostaining Mosaic analysis with a repressible cell marker Multicolor celllabeling
We thank J. Goedhart for sharing the mTurquoise and mTurquoise2 cDNA. D. Hadjieconomou developed the original and C sets of FB transgenes. N. Shimosako generated the B set of transgenes and validated the hs-mFLP5MH insertions. The original FB approach was developed in collaboration with B.J. Dickson, S. Rotkopf, C. Alexandre, and D.M. Bell. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001151), the UK Medical Research Council (FC001151), and the Wellcome Trust (FC001151), and by the UK Medical Research Council (U117581332).