Zebrafish were maintained and raised in our facility under recommended conditions . The following strains of fish were used in this study: AB (wild type), casper , was , cldnB::GFP , BACmpx::GFP , Brn3c::mGFP , Tg(lyz:EGFP)nz117 and Tg(lyz:DsRED2)nz50, herein named lysC::GFP and lysC::DsRED2 . All embryos were collected by natural spawning, staged according to Kimmel et al.  and raised at 28°C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% methylene blue, equilibrated to pH 7.0) in Petri dishes, as described previously . Embryonic and larval ages are expressed in hours postfertilization (hpf). All animals subjected to experimentation were anesthetized in MS-222 (tricaine; A5040; Sigma, Saint Louis, MO, USA), and procedures complied with the guidelines of the Animal Ethics Committees of the University of Chile and Karlsruhe Institute of Technology.
A 10 mM stock solution of CuSO4 (copper II sulfate pentahydrate, catalog no. 102780; Merck, Darmstadt, Germany) was prepared daily in bidistilled water in a glass beaker until dissolved completely. Likewise, CdCl2 (CB236; Matheson, Coleman & Bell, Cincinnati, OH, USA) was prepared at a stock concentration of 10 mM. Additional chemicals were ZnSO4 (catalog no. ZI-1705; Winkler S.A., Santiago, Chile), NiSO4 (72280; Sigma) and AgNO3 (101512; Merck), neomycin (N1876; Sigma), 3,3'-dihexyloxacarbocyanine iodide, or DiOC6(3), was purchased from Interchim, Montluçon, France (FP-46764A) or AnaSpec, Fremont, CA, USA (catalog number 84715), DPI (D2926; Sigma), DMSO (317275; Merck), paraformaldehyde (catalog no. 1.04005.1000; Merck), Sudan Black (380B; Sigma), and Tween 20 (P5927; Sigma).
The following anti-inflammatory drugs were tested: from Cayman Chemical Co. (Ann Arbor, MI, USA), ibuprofen (70280), diclofenac (70680), aspirin (70260), indomethacin (70270), trans -resveratrol (70675), rosiglitazone (71740), mifepristone (10006317), sulindac (10004386) and SP600125 (10010466). From Sigma-Aldrich (St. Louis, MO, USA): dexomethazone (D1756) and hydrocortisone (H4001).
Neuromast damage protocol and the basic ChIn assay (manual quantification)
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 Figure 2) 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 , 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 . AB zebrafish were used as wild-type controls.
All drugs were prepared according to the manufacturer's instructions at a stock concentration of 10 mM by dissolving in 100% DMSO, which was previously purged by gaseous nitrogen for 2 minutes. Drugs were stored at -20°C until use and were diluted immediately prior to being added to larvae medium.
Drugs were added to the required concentration into the wells containing the experimental larvae in E3 containing 1% DMSO. Positive and negative control larvae were incubated only with 1% DMSO. Incubation with drugs was done for 1 hour prior to addition of CuSO4, which was added directly to the wells containing the experimental and positive control larvae. Incubation after copper addition continued for another 40 minutes before fixation in 4% paraformaldehyde for 1 hour at ambient temperature in the dark. Counting of leukocytes was carried out as before.
Data are presented as mean values ± SEM. Statistical analysis was performed using GraphPad Prism version 5.00 for Windows software (GraphPad Software, La Jolla, CA, USA). The probability level for statistical significance was P < 0.05. All statistics regarding leukocyte migration were evaluated with unpaired t -tests with Welch's correction.
For imaging, larvae were anesthetized and mounted in 1.5% low melting point agarose (peqGOLD 35-2099; PEQLAB Biotechnologie, Erlangen, Germany) dissolved in E3. Photographs were taken with a Leica DFC 300-FX camera and Leica SPE confocal microscope (Leica Microsystems, Wetzlar, Germany) and processed with Adobe PhotoShop (San José, CA, USA), Zeiss Axiovision (Carl Zeiss Microimaging GmbH, Jena, Germany) and Image J (version 4.2, http://rsbweb.nih.gov/ij/index.html) software. For time lapse imaging, we used a Zeiss Axiovert 200 M microscope equipped with a ×20 lens objective and an Axiocam camera (Additional file 2) or a Leica SPE confocal microscope using a ×40 lens objective (Additional file 3). As described in Additional file 2 larvae were incubated in a 1:3,000 dilution of DiOC6(3) stock solution (1 mg/ml) in E3 and were then washed three times with E3 for 5 minutes. Larvae were embedded in 1.5% low melting point agarose dissolved in E3 containing 50 μM CuSO4. Images were captured every 90 seconds for a total of 150 minutes. As described in Additional file 3 compound transgenic fish (cldnB::GFP, lysC::DsRED2) were treated with 50 μM CuSO4 for 5 minutes and then mounted in 1.5% low melting point agarose dissolved in E3 containing 50 μM CuSO4. Images were captured every 60 seconds for a total of 100 minutes.
Automated ChIn assay
Individual anesthetized larvae were manually placed in single wells of a 384-well plate in embryo buffer. Subsequently, compound stock solution in DMSO was transferred from a compound source plate to the assay plate containing embryo buffer (E3 + 1% DMSO + 0.02% MS222) and larvae using a multichannel pipette. The assay plate containing larvae and compound was sealed and incubated for 30 minutes at 28°C. Following compound incubation, CuSO4 solution (0.3 mM) was added to the assay plate, resulting in a final concentration of 10 μM copper sulfate. Assay plates were then incubated for another 40 minutes at 28°C. This procedure yielded a total volume of 120 μl per well and resulted in the respective final compound screening concentrations for this assay (10 μM ibuprofen, 3 μM diclofenac, 10 μM indomethacin, 1 mM dexamethazone, 300 μM hydrocortisone). A volume of 120 μl was empirically identified as the optimal volume for our automated imaging procedure. To achieve an assay sensitivity comparable to manual analysis, we also defined the minimum number of individual larvae analyzed per condition to be 30. In practice, 24 embryos for one condition were imaged per 384-well plate, but experiments were repeated at least in triplicate such that, on average, 50 larvae were analyzed per condition (see below).
Automatic imaging was performed on Olympus Scan^R high-content screening microscope setups (Olympus Biosystems, Munich, Germany) equipped with a ×2.5 lens objective (plan-apochromatic), an Olympus Biosystems DB-1 digital camera (1,300 × 1,024 pixels), filter cubes for GFP excitation filter, 460-480 nm; emission filter, 495-540 nm; dichromatic mirror, 485 nm) and cyanine 3 (Cy3) (excitation filter, 535-555 nm; emission filter, 570-625 nm; dichromatic mirror, 565 nm), and an ultrastable light source MT-20 xenon lamp. Camera image integration times were fixed (20-ms bright-field microscope, 400-ms GFP, 150-ms Cy3). An object detection autofocus algorithm detected the central focal plane of the first-well larva and was applied for the rest of the plate. Image processing was done using the LabView Vision AI rapid prototyping tool (National Instruments, Munich, Germany). Data management, red-green-blue (or RGB) overlay gallery generation and result display were performed using self-made LabView software modules. These scripts enable the detection of GFP-labeled neuromasts on an extended focus projection of five optical sections (two in each direction from the central focal plane) and define an empirically established surrounding area (see Figure 4) in the GFP channel. Subsequently, the inflammatory response is quantified by the detection of DsRED2-labeled leukocytes within the area surrounding the neuromasts in the red channel. Quantification is based on the average relative fluorescence intensity of leukocytes within this area. For proper image processing, the larvae ideally have to be oriented in a lateral position. However, our image analysis scripts allowed a certain degree of freedom with regard to this requirement. Our procedures yielded more than 70% of larvae positioned in a way that allowed automated image processing without the need for manual orientation of the larvae. However, all plates were manually checked, and larvae in unfavorable positions were corrected. All modules are available on request from the authors.