Mz16f microscope

NF-48 tadpoles treated and fixed as for morphology, were stained with Alcian blue to visualize cartilage based on guidelines for whole mount Xenopus [59 (link)]. In brief, fixed tadpoles were bleached overnight in 50% ethanol/3% hydrogen peroxide and then transferred through an ethanol series up to 100% ethanol. Dehydrated tadpoles were stained for 2–3 h in 0.2% Alcian blue 8GX (Sigma-Aldrich, A5268)/70% ethanol/30% glacial acetic acid. Samples were washed 2–3 times in 70% ethanol/30% glacial acetic acid, and then allowed to destain over night. Tadpoles were rehydrated through a decreasing ethanol series, then cleared in 0.25% trypsin/30% saturated borax for 30 min at room temperature, and then stored at 4°C in PBS/0.02% sodium azide. Alcian blue stained tadpoles were photographed using a Leica MZ16F microscope and a Leica DFC 500 camera.

Using a Leica M2 16 F fluorescent stereomicroscope, the embryos were imaged in system water (0.8 g sodium bicarbonate, 4.5 mL Marine Salts, 750 L H₂0, 0.6 mL methylthioninium chloride [methylene blue]) with 4.2% (v/v) Tricaine to induce anaesthesia. Images were taken at 40 x, 80 x or 100 x magnification using a Leica DFC300 FX Digital Colour Camera connected to LAS AF (Leica) V3 software connected to the Leica MZ 16 F microscope. For confocal imaging (Leica sp5), the embryos were mounted in 1.5% low melting-point agarose (Sigma Aldrich).

Anti-sense WISH probes for Pierce1 and Pierce2 were generated against DNA sequences corresponding to Pierce1 16-768 nt (NM_027040.1) and Pierce2 98-550 nt (NM_001198789.1). PCR-generated sequences were ligated into the pBluescript II KS(−) vector linearized with EcoRV. The identity of the cloned sequence was confirmed by DNA sequencing (Source BioScience) primed using the T3 and T7 promoter sequences. Digoxygenin-labeled anti-sense riboprobes for Cerl2 (Marques et al., 2004 (link)), Pitx2 (Ryan et al., 1998 (link)), Pierce1, and Pierce2 were transcribed from either the T3 or T7 promoter. WISH experiments were performed as described (Field et al., 2011 (link)) using anti-Digoxigenin antibody (Roche, #11093274910) and NBT/BCIP staining (Roche, #11681460001). Stained embryos were imaged in PBS using a Leica DFC420 camera on a Leica MZ16F microscope. Whole embryos were taken after imaging for genotyping.

Zebrafish studies were completed under the approval of the University of Liverpool Animal Welfare and Ethical Review Body. Nacre ubiq:secAnnexinV-mVenus embryos^{24 (link)}, obtained from The University of Manchester Biological Services Facility, were incubated at 28 °C in egg water (60 μg/ml Tropic Marin salt in distilled water) until 48 h postfertilization (hpf). Following cell implantation, fish were maintained in a humidified light-cycling incubator (14 h on, 10 h off) at 34 °C. For toxicity studies, embryos at 72 hpf were exposed to increasing concentrations of BH3 mimetics (at 34 °C) and mortality assessed up to 120 hpf. For xenograft studies, UM-SCC-81B cells stably expressing H2B-mRFP were injected into the pericardial cavity at 48 hpf, as previously described^{25 (link)}. At 72 hpf, fish were screened for the presence of red fluorescent cell masses, following anesthetization using MS-222 (160 µg/ml for maximum 15 min) and images acquired using a Leica MZ16F microscope. Successfully xenografted fish were randomly placed individually in wells of a 24-well plate and either DMSO or BH3 mimetics added to 1 mM Tris-buffered egg water. Fish were euthanized by 120 hpf using MS-222 (250 µg/ml). Images of tumor masses were acquired and 2D tumor areas calculated using ImageJ software.

Distal mesenchyme or polarizing region tissue from replicate experiments (14 wing bud samples from 7 embryos) was dissected into 100 μm blocks in ice cold PBS under a Leica MZ16F microscope using a fine surgical knife and pooled. Blocks were digested into single cell suspensions with 0.5% trypsin (Gibco) for 30 min at room temperature. Cells were briefly washed twice in PBS, fixed in 70% ethanol overnight, washed in PBS and resuspended in PBS containing 0.1% Triton X-100, 50 µg ml⁻¹ propidium iodide and 50 µg ml⁻¹ RNase A (Sigma). Dissociated cells were left at room temperature for 20 min, cell aggregates were removed by filtration and single cells analysed for DNA content with a FACSCalibur flow cytometer and FlowJo software (Tree Star). Based on ploidy values, cells were assigned to G1-, S- or G2/M-phase and this was expressed as a percentage of the total cell number (approximately 5000 in each case). Statistical significance of numbers of cells between pools of dissected wing bud tissue (14 in each pool) was determined by Pearson's χ² tests to obtain two-tailed P-values [significantly different being a P-value of less than 0.05 (Chinnaiya et al., 2014 (link)) for statistical comparisons of cell cycle parameters between the wing buds of embryos incubated together].