Computational protocol: Phosphatidylserine save-me signals drive functional recovery of severed axons in Caenorhabditis elegans

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Protocol publication

[…] The maximum intensity of tagRFP and UNC-104::GFP was calculated 6 h postaxotomy from a region at the end of both the proximal and distal sides of the cut site using ImageJ 1.47n software. GFP intensity was normalized to tagRFP, and these values were used to compare the relative GFP intensity on the proximal and distal cut sites. GFP intensity 24 h postaxotomy was quantified from measurements taken at the fusion site or at the end of the longest regrowing branch before being compared with the distal side of the cut site. “No cut” control animals were imaged without axotomy, and measurements were calculated ≈50 μm anterior to the PLM cell body. [...] To induce sAnx::mRFP expression, smIs95(Phsp16-2::sAnxV::mRFP); zdIs5 animals were incubated at 30 °C for 30 min 4 h before analysis. PS exposure was visualized with a Leica SP8 confocal microscope equipped with LAS X software. Green fluorescence was visualized with a 488-nm laser (2.4% power; 600 gain; 8× averaging), and red fluorescence was visualized with a 543-nm laser (100% power; 1,000 gain; 8× averaging). Fluorescence intensities were calculated from line scans recorded in 15-μm measurements along both the proximal and distal axon segments using ImageJ 1.47n software. To avoid fluorescence caused by collateral damage, line scans were taken 5 μm away from the cut site. mRFP expression 1 h postaxotomy was calculated relative to expression levels immediately preceding axotomy. […]

Pipeline specifications

Software tools ImageJ, LAS X
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Caenorhabditis elegans