2 Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction 39 Evaluation of locomotor activity Fivedpf ctns‒/‒ and wt zebrafish larvae were preincubated in 100 µl of 0.3× Danieau’s solution (1.5 mM HEPES, pH 7.6, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, and 0.18 mM CaNO32) in individual wells of a 96-well plate at 28.5°C (N=56 and 52, respectively). Larvae were allowed to habituate for 10 min in the light in a chamber of an automated tracking device (ZebraBoxTM; Viewpoint, Lyon, France) followed by 1h tracking in the light, then 10 min habituation in the dark followed by 1h tracking in the dark. Locomotor activities of two independent experiments were quantified using ZebraLabTM software (Viewpoint, Lyon, France). Total movement or activity was expressed in ‘‘actinteg’’ units reported every 5 min of the tracking period62. The actinteg value of the ZebraLabTM software is defined as the sum of all image pixel changes detected for each larva during the reporting time period. Microscopy Light microscopy: whole zebrafish larvae at 3 and 6 dpf were prepared for light microscopy. They were fixed in 4% PFA for 24 hours, transferred to 70% ethanol and embedded in paraffin. After transversal sectioning (3 µm), the samples were deparaffinised and stained with haematoxylin and eosin. Block face scanning electron microscopy: Samples were prepared and imaged with the help of the EM Facility at the Faculty of Life Sciences, University of Manchester, UK, as previously described38. Four dpf wt and ctns‒/‒ larvae were fixed in 2.5% glutaraldehyde/4% formaldehyde in 0.1 HEPES, pH 7.2, before high density staining for serial block face imaging. Briefly, samples were washed in ddH2O and incubated in 1% osmium tetroxide/1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 1 hour at RT and washed again. Specimens were then incubated for 1 hr at RT in 1% aqueous thiocarbohydrazide, then 1 hr incubation in 1% aqueous osmium tetroxide followed by 1 hr incubation in 1% aqueous uranyl acetate. Samples were then incubated at 60°C for 30 mins in Walton’s lead aspartate solution, followed by dehydration in a graded ethanol series and acetone. Samples were embedded in TAAB 812 Hard and trimmed to locate the pronephros. Serial block face scanning EM was carried out using a Gatan 3View microtome within an FEI Quanta 250 FEG scanning electron microscope. Large images were acquired following ten 100 nm cuts of pronephros (54.4 mm x 54.4 mm) with a pixel resolution ~10 nm and 10 ms dwell time. Image analysis for lysosomal number and surface area was performed using ImageJ software. Transmission electron microscopy: wt and ctns‒/‒ larvae at 6 dpf were fixed with 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 24 hours, rinsed twice with 0.1 M sodium cacodylate, incubated for 1 hour in 1% osmium tetroxide in 0.1 M sodium
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