MS-222

Embryos from wildtype (TL, AB/TU, and WIK), transgenic (Tg(lyz:EGFP)[28 (link)], Tg(mpeg1:EGFP)[29 (link)], and Tg(pu.1:Gal4-UAS-EGFP)[22 (link)]), and irf8^st95 and irf8^st96 heterozygous intercross backgrounds were raised at 28.5°C, and staged by established standards [40 (link)]. Embryos were treated with 0.003% 1-phenyl-2-thiourea (PTU) in methylene blue embryo water to inhibit pigmentation. For in situ hybridization and TUNEL staining, zebrafish embryos and larvae were fixed immediately at the indicated time points of analysis using 4% paraformaldehyde/PBS for overnight fixation at 4°C. For fluorescent imaging, zebrafish embryos and larvae up to 7 dpf were imaged in the living animals at the time of analysis, and older larvae up to 31 dpf were imaged after fixation; they were briefly anesthetized using 0.02% MS-222 (tricaine) prior to overnight fixation in 4% paraformaldehyde/PBS. All euthanasia and procedures followed the protocols approved by the Stanford Institutional Animal Care and Use Committee.

We implanted electroencephalogram (EEG) electrodes for recording brain activity in four wild-caught adult female cane toads (115–133 mm snout-urostyle length, 201–235 g). After the animals were anaesthetised with MS 222 (tricaine methanesulfonate; 3 g/l), we drilled four holes (0.5 mm diameter, two per cerebral hemisphere) through the exposed cranium to the level of the dura overlying the dorsal cortex. A fifth hole was drilled over the olfactory bulbs for the ground. All electrodes were gold-plated, round-tipped pins (0.5 mm diameter) glued in place using cyanoacrylic adhesive. Electrode wires (AS633, Cooner Wire, Chatsworth, California, USA) terminated at a connector fixed on the head with two stainless steel screws (25/095/0000, Hilco, London) and light-curing dental acrylic (Dentsply, Mt Waverley, Victoria). Electrode position was verified by dissection at the end of the study. Toads were then transferred to a damp cage, monitored until they regained normal motor function, and allowed a 10-day period of post-operative recovery at 30°C with food and water available ad libitum. EEG activity was recorded at 100 Hz using a head-mounted, miniature (25×25×9 mm) and lightweight (8 g, including battery) Neurologger 2A datalogger (Vyssotski et al., 2009 (link); Lesku et al., 2012 (link)).
To record body temperatures during cooling and freezing, we inserted a thermocouple wire subdermally into each toad's left hind limb (to measure temperature immediately below the skin) and into the cloaca (to measure deep body temperature), respectively. These thermocouple leads, as well as one measuring ambient temperature, were connected to a TC-2000 thermocouple meter (Sable Systems, Las Vegas, NV USA) and logged each minute using ExpeData software via a UI2 analogue/digital converter temperature logger (Sable Systems, Las Vegas, NV USA). Prior to experiments, toads were transferred into a Faraday cage (22×17×12 cm) then placed into a standard household refrigerator (Kelvinator, Charlotte, NC USA). Once the toad's core reached fridge temperature (∼5°C), it was transferred to a household freezer (Fisher and Paykel, Auckland, New Zealand) for 30 min. Toads removed from the freezer after this time were dead (did not regain consciousness).
Fast Fourier Transforms were performed on 4-s, artefact-free epochs to calculate power in 0.39 Hz bins between 1.17 and 49.61 Hz using RemLogic 3.2 software (Embla Systems, Broomfield, CO USA). Cumulative EEG power was calculated as a measure of brain activity in 10-min bins, starting at the time placed in the new thermal regime. We also quantified power in the bandwidths typically used in analysis of the mammalian EEG (delta, theta, alpha, beta and gamma) and recently applied to amphibians (Fang et al., 2012 (link)).
All procedures were approved by the University of Wollongong Animal Ethics Committee (protocol no. AE10/05).

Lines included in our database were crossed to vglut2a:DsRed, which broadly labels glutamatergic neurons throughout the brain (Kinkhabwala et al., 2011 (link); Satou et al., 2012 (link)), providing a suitable channel for image registration. Embryos from these crosses were raised in E3h media containing 300 μM N-Phenylthiourea (PTU) starting at 8–22 h post fertilization (hpf) to suppress melanophore formation and sorted for fluorescence at 2 days post fertilization (2 dpf). An inverted laser-scanning confocal microscope (Leica TCS SP5 II) equipped with an automated stage and 25x/0.95 numerical aperture apochromatic water immersion lens (Leica # 11506340) was used to acquire confocal stacks of transgenic fish and immunofluorescently labeled samples. Live larvae were anesthetized in 0.24 mg/mL tricaine methanesulfonate (MS-222) for 3 min prior to mounting at 6 dpf. Live or fixed embryos were then mounted in 2.5% low melting point agarose placed in a 3D printed ABS four-well plastic insert (Stratasys uPrint) within a cell culture chamber with a number 1.5 thickness (0.17 ± 0.005 mm) cover glass bottom (Lab-Tek II 155379). A 488 nm argon laser line and a 561 nm diode-pumped solid state (DPSS) laser were used to excite fluorophores with images acquired as serial sections along the z-axis at 2.0 μm intervals between dorsal and ventral surfaces of the brain in a 1 × 2 tiled array to visualize the brain and a portion of the spinal cord. To shorten scan duration and allow for laser compensation over the z-dimension, channels were acquired simultaneously at a 1 × 1 × 2 μm; xyz pixel spacing. Laser illumination was increased via acousto-optic tunable filter with z-position to counter attenuation with ramp points set every ~70 μm. Fluorescent emission from the 488 nm excitation was collected with a hybrid detector with a spectral window of 500–550 nm and emission from the 561 nm excitation collected by a second hybrid detector set at 571–700 nm. In order to minimize the cross-channel contamination present between imaged fluorophores, dye separation was performed in the Leica acquisition software (Leica Application Suite—Advanced Fluorescence, LAS AF) with coefficients based on published spectra of imaged fluorophores and the spectral windows used for acquisition. Resulting rostral and caudal stacks were stitched together (Preibisch et al., 2009 (link)) and channels split in Fiji (Schindelin et al., 2012 (link)) prior to registration.

The effects of dietary EPA and DHA concentrations on flesh pigmentation were evaluated when the fish were 1200 g and 3.5 kg. At 1.2 and 3.5 kg, the fillet (right side) was sampled from 30 fish per diet treatment and stored on −80°C for later analysis of carotenoid and fatty acid composition as described below and in Bou et al., [3 (link)]. Fish were killed by an overdose of the anesthetic metacain (MS-222; 0.08 g/l), and individual weights and lengths were recorded. The skin and bone of the fillet were removed before the fillet was homogenized, and the carotenoids extracted using a 1: 1 : 3 mixture of distilled water, methanol (containing 500 ppm butylated hydroxytoluene), and chloroform as described by Bjerkeng et al., [33 (link)]. A Spherisorb S5CN-4800 nitrile column (Hichrom Ltd., Theale, Berkshire, UK) was used to determine the amount of astaxanthin and idoxanthin in the samples using a mobile phase with 20% (v/v) acetone in n-hexane+ (HPLC system I). An external standard of all-E-astaxanthin (Hoffmann-La Roche, Basel, Switzerland) with known concentration was prepared to establish a response line, and sample concentrations were calculated using peak areas from the chromatograms. The concentration of astaxanthin standards were determined spectrophotometrically in n-hexane containing 4.5% (v/v) CHCl₃. Standards of idoxanthin 3′, 4′-cis and trans glycolic isomers were prepared according to Aas et al. [29 (link)]. The retention times (R_T) for the 3′, 4′-cis and 3′, 4′-trans glycolic isomers of idoxanthin were ca. 7.6 and 9.3 min, respectively.

As the cells reach the horizontal myoseptum, the segmental artery tip cell undergoes a single cell division (20.5–23.5 hours post-fertilization (hpf)), and after this, one cell maintains its position and becomes a connector cell, while the tip cell continues to migrate dorsally, forming dorsal longitudinal anastomotic vessel (DLAV, 30–31 hpf). By 2 hpf, the trunk and tail intersegmental vessels (ISVs) for most zebrafish are lumenized and have an active circulation [23 (link),24 (link)].
In order to evaluate the formation of blood vessels in zebrafish embryos, 22 hpf embryos were distributed in 12-well plates (30 embryos per well) (BD Falcon) for a treatment period of 26 h. The positive control for this assay was 5 μM PTK787, which is a VEGFR antagonist [25 (link)], and the negative control was 0.1% dimethyl sulfoxide (DMSO). Different concentrations of apatinib (1, 2.5, 5, and 10 μM) and PTK787 (5 μM) were diluted in 0.1% DMSO. DMSO containing the drug of interest was soaked in fish water, and zebrafish could take the drug orally. After treatment, the embryos were anesthetized with 0.016% MS-222 (tricaine methanesulfonate, Sigma-Aldrich, St. Louis, MO) and the number of complete ISVs (i.e., the number of ISVs that connect the dorsal aorta (DA) to the DLAV) was counted. The drug effect was then calculated using the following formula: $\%\text{Inhibition}=\left(1-\frac{\text{ISV amount of experimental group}}{\text{ISV amount of vehicle control}}\right)\times 100.$

Live fluorescence imaging was performed using a stereomicroscope (Nikon SMZ18) equipped with the NIS-Elements BR 3.0 software or confocal microscopes (Zeiss Meta 500; Olympus FluoView FV3000; Zeiss LSM900). Medaka hatchlings (8 to 23 dpf) were anaesthetized with 0.005% ethyl 3-aminobenzoate methane sulfonate (Tricaine; Sigma MS-222) and mounted in 1.5% low-melting-point agarose on a glass bottom petri dish. Confocal pictures were taken using 405, 488, 543 or 633 nm laser lines for CFP, GFP, mCherry and Cy5 fluorescent signals, respectively. Time-lapse imaging was performed with Olympus FV3000 or Zeiss LSM900 microscopes by imaging the region of interest for 15-20 hours with 5-10 mins intervals. Imaging data were processed using Olympus FV31S-SW 2.1.1.98, Bitplane Imaris 9.0, ImageJ and Adobe Photoshop CC 2018 software.