Zebrafish larvae of the BACmpx::GFP and lysC::DsRED2 strains were grown in E3 medium in groups of 40-50 larvae per 10-cm Petri dish until 56 hpf. Only those larvae that spontaneously hatched were used for the assays as artificial enzymatic dechorionation often damages neuromasts, causing spontaneous inflammation. Any fish that appeared developmentally delayed or otherwise abnormal were also excluded from further analysis.
Selected larvae were transferred to six-well plates (M8562; Sigma) in a volume of 6 ml of E3 solution lacking methylene blue, and 15 larvae were added per well. Stock solutions of CuSO4 were added directly to the wells, and incubation was carried out for 40 minutes at 28°C. Larvae were then fixed by transferring them to 1.5-ml microfuge tubes and replacing the E3 medium with 4% paraformaldehyde prepared in phosphate-buffered saline (PBS) and incubating for 1 hour at room temperature. During fixation and subsequent handling, the tubes were kept in the dark to avoid bleaching or fading of the fluorescent protein signal. After fixation, larvae were washed three times for 5 minutes each in PBS-Tween20 with gentle agitation. Examination of fluorescent cells and counting was carried out within the next 48 hours after fixation using a Leica (Wetzlar, Germany) MZ-12 fluorescent stereoscope. Labeled cells were counted under fluorescent illumination within 10 cell diameters of the horizontal myoseptum between the first somite and the end of the tail (see Figure2 ) on one side of each larva. All experiments were carried out with a minimum of 15 larvae for each condition, and counts were carried out by two observers. For neomycin treatments, BACmpx::GFP fish were incubated in the indicated concentration of antibiotic for 1 hour, and cell counts were done as before. In these experiments, we used 96-hpf fish, as neomycin kills only mature hair cells in lateral line neuromasts. We confirmed cell death in these fish by using hair cell-specific markers.
For Sudan Black staining, we used fish of the casper mutant strain [34 (link)], which lack pigmentation in the body. Larvae at 56 hpf were incubated as before (no metal and 10 mM CuSO4), fixed, washed and incubated for 20 minutes in 0.5 ml of Sudan Black staining reagent in batches of 30 larvae. Larvae were then washed three times in 70% ethanol at room temperature with mild rocking. Labeled cells were counted as before under bright-field illumination under a dissecting stereoscope. The was mutant fish were kept in E3 supplemented with propylthiouracil (from 24 hpf until fixation) to suppress pigmentation. The was+/- fish were mated with was-/- fish. Entire clutches were scored prior to genotyping, which was carried out as described previously [7 (link)]. AB zebrafish were used as wild-type controls.
Selected larvae were transferred to six-well plates (M8562; Sigma) in a volume of 6 ml of E3 solution lacking methylene blue, and 15 larvae were added per well. Stock solutions of CuSO4 were added directly to the wells, and incubation was carried out for 40 minutes at 28°C. Larvae were then fixed by transferring them to 1.5-ml microfuge tubes and replacing the E3 medium with 4% paraformaldehyde prepared in phosphate-buffered saline (PBS) and incubating for 1 hour at room temperature. During fixation and subsequent handling, the tubes were kept in the dark to avoid bleaching or fading of the fluorescent protein signal. After fixation, larvae were washed three times for 5 minutes each in PBS-Tween20 with gentle agitation. Examination of fluorescent cells and counting was carried out within the next 48 hours after fixation using a Leica (Wetzlar, Germany) MZ-12 fluorescent stereoscope. Labeled cells were counted under fluorescent illumination within 10 cell diameters of the horizontal myoseptum between the first somite and the end of the tail (see Figure
For Sudan Black staining, we used fish of the casper mutant strain [34 (link)], which lack pigmentation in the body. Larvae at 56 hpf were incubated as before (no metal and 10 mM CuSO4), fixed, washed and incubated for 20 minutes in 0.5 ml of Sudan Black staining reagent in batches of 30 larvae. Larvae were then washed three times in 70% ethanol at room temperature with mild rocking. Labeled cells were counted as before under bright-field illumination under a dissecting stereoscope. The was mutant fish were kept in E3 supplemented with propylthiouracil (from 24 hpf until fixation) to suppress pigmentation. The was+/- fish were mated with was-/- fish. Entire clutches were scored prior to genotyping, which was carried out as described previously [7 (link)]. AB zebrafish were used as wild-type controls.
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