Computational protocol: High-resolution in vivo imaging of regenerating dendrites of Drosophila sensory neurons during metamorphosis: local filopodial degeneration and heterotypic dendrite–dendrite contacts

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Protocol publication

[…] To acquire images of da neurons at pupal stages, the collected pupae were transferred to a plate filled with water, washed and dried on 3MM Chr paper (Whatman). Each pupa was taken out of its puparium carefully by forceps, mounted in PBS on a slide between spacers made of vinyl tape, and covered with a 24 × 24 mm cover slip (No. 1, MATSUNAMI). For imaging, adult abdomens, heads, wings and legs were removed and mounted in 50% glycerol as described above. Images of ldaA/A-like and v'ada neurons at A4 and/or A5 were acquired by laser scanning confocal microscopy (NikonC1) with 488- or 543-nm lasers with a 1-μm Z-step and total 15–20 μm depth. Images were processed by using Photoshop, Illustrator (Adobe Systems), and ImageJ. Neurocyte software (Kurabo) was used for the quantification of total dendritic length. Statistical analysis was performed by R program (version 2.14.0; The R Foundation for Statistical Computing). [...] Each pupa was taken out of its puparium as described above and mounted on a 35-mm glass-bottomed dish (3911-035, IWAKI). In mounting, we folded legs and put abdomens on the dish and tilted them to retain an appropriate angle to observe v'ada and/or ldaA/A-like neurons. Details of image acquisition are essentially described previously (), except for (Supporting Information). Briefly, – (Supporting Information) were acquired by using a spinning-disk confocal scan head (CSU10; Yokogawa), an Olympus IX71 microscope and an EM-CCD camera (DU-888; Andor Technology). Fluorescent proteins were excited with a 488-nm line and a 561-nm line, and signals were detected with a 500- to 550-nm and 580- to 640-nm band-pass filter, respectively. The exposure time was 500 ms for both 488 and 561 nm, and the EM gain of the camera was 1000×. The output of the 488- and 561-nm laser power was 10% and 40%–50%, respectively. Typically, single confocal planes were taken with a 0.7-μm Z-step and total 15–20 μm depth, at 30-s or 60-s intervals. The above hardware was driven by MetaMorph (Molecular Device), and acquired data were processed with MetaMorph and ImageJ. After image acquisition, each pupa was kept at 25 °C and their survival was confirmed to at least the pharate stage or the adult stage. (Supporting Information) was acquired by using a Leica TCS SPE (see its legend). […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Drosophila melanogaster
Diseases Keratitis, Dendritic