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Fig. 1
405 nm laser ablation induces kidney specific segmental epithelial injury in GFP transgenic zebrafish.
(A) 12 dpf zebrafish was subjected to 405 nm laser treatment using a confocal microscope. The plane of maximal illumination was guided by kidney GFP fluorescence (here- E11-9 transgenic fish). The laser scan window is shown by the white rectangle. Efficient laser treatment was monitored by observing GFP bleaching in the ablated area (with a target of ~50% initial reduction in GFP fluorescence). The ablated segment continued to lose GFP positivity with only an occasional cell surviving at 160 min post- laser treatment. The arrow points to a sharply defined edge of surviving epithelium. Scale bar = 70 μm. (B) Propidium iodide (PrI, red) staining confirmed that loss of GFP positivity was due to cell death, as opposed to other mechanisms. As cells in the injured segment lost GFP positivity, they became strongly PrI-positive. Every cell showed PrI positivity by 60 min post injury, and by 210 min PrI-positive material was seen exclusively in the distended lumen proximal to the obstructed segment (lower panel). PrI-positive material could also be seen transiting through the distal tubule (arrow in 20 min panel). Scale bar = 100 μm. (C) Electron microscopy of the injured epithelium 3 hours post-laser treatment shows compacted swollen degenerated mitochondria (arrowheads). The basement membrane (pink, arrows) and the adjacent, likely stromal or endothelial cell (green, star) is preserved. “n” – intact nucleus, “ly” – lysosome. Scale bar = 1000 nm.