Tricaine ms 222

The quantification of mean fluorescence intensity (MFI) indicated kidney inflammation. Tg(lysC:DsRed2)^nz50 larvae were anesthetized with 0.168 mg/mL of Tricaine (MS222) (Sigma-Aldrich, St. Louis, MO, USA) and placed on a microscope slide with 3% methylcellulose [56 (link),57 (link)]. The animals from the control and treated groups were carefully manipulated for lateral vision, and images were captured using the ZEISS Axio Zoom.V16. The MFI of neutrophils in the glomerular region of the pronephros was quantified using ImageJ software.

Fish were sedated with 200 μg/ml ethyl-3-aminobenzoate methanesulfonate (Tricaine/MS222; Sigma; A5040) in system water buffered to pH 7.0–7.5 with NaHCO₃ (Sigma; S5761) and imaged on moist filter paper with an MZ16 FA fluorescence stereomicroscope (Leica, Wetzlar, Germany) and DFC 300 FX Digital Color Camera (Leica) at 1.4 MPixel resolution at 7.11× and 14× magnification (55 ). Images were stitched together in Pixelmator 3.5 Canyon software (Pixelmator Team, Vilnius, Lithuania). Total body length was measured using Fiji software, and differences in mean total body length between genotypes were tested for significance by two-sided Student’s t-test. The number of fish with dorsal tilting of the head was analyzed by the Fisher Exact test in SPSS software version 22 (IBM, Armonk, NY, USA).

Embryo tracking on fresh 1% agarose gels was performed using the Serial Images function of AxioCam MRc 5 microscope (Zeiss). 10 embryos per condition were transferred to 0.02% Tricaine/MS222 (Sigma) for anesthesia before they were placed on a 1% agarose gel where 20 images were taken, one image per sec. FIJI software was used to generate time-lapse movies of 5 frames/sec. Embryos were tracked with the ‘manual tracking’ function and the distance traveled was calculated as mm/min for each embryo in Microsoft Excel. For rescue experiments, the same embryos were tracked before and after treatment with DMSO/OA. Four embryos were chosen as representative images per figure for each condition and the background was adjusted using the Magnetic Lasso tool in Adobe Photoshop CS6. Quantifications were performed for the indicated number of embryos from 3 or more independent injections.

Six to twelve animals at the age of 8 weeks, 20 weeks, 36 weeks and 54 weeks were anesthetized with Tricaine/MS-222 (0.2 mg mL⁻¹; Sigma-Aldrich, Munich, Germany), and 50% of the caudal fin was amputated with a scalpel. Fins of individual fish were photographed using the stereo microscope Lumar V12 (Zeiss, Jena, Germany) directly after amputation and every second day post amputation including the first day for the duration of 27 days. Outgrowth length of concentric fin rays was determined for every time point in pixels and converted into millimetre using Photoshop (Adobe, San Jose, CA, USA). Next, the so-determined outgrowth length was related to the original length of the amputated fin. The regenerative growth rate was determined by the increase in outgrowth length between two time points in relation to the length of the original amputated fin. Significant differences between age groups were analysed at each time point using one-way ANOVA followed by pairwise comparison using Tukey’s HSD test.

Commercially available red palm oil (RPO) was purchased from a local supplier in Malaysia. RPO is non-GMO-certified and contains 664.994±1.946 ppm (0.44:0.56, α-carotene: β-carotene) of carotenoids and 999.485±20.023 ppm of vitamin E, respectively. Polyoxyethylenesorbitan monooleate (Tween 80), sorbitan monoolete (Span 80) and glycerol were obtained from SystermChemAR (Shah Alam, Malaysia). The water used was Ultrapure water from Milli-Q Plus. For high-performance liquid chromatography (HPLC) analysis, isopropanol, hexane, methanol, acetonitrile, ethyl acetate, triethylamine and 1,4-dioxane were purchased from Fisher Scientific (Loughborough, United Kingdom). Ascorbic acid and DPPH (2, 2 diphenyl-1-picryl hydrazyl) were obtained from TCI America (Tokyo, Japan), while ethanol was obtained from Merck (Darmstadt, Germany). Primary epidermal keratinocytes (normal human, adult, PCS- 200–011) and complete growth medium were purchased from American Type Culture Collection (Virginia, ATCC, USA). Tricaine (MS-222) was purchased from Sigma. Dimethyl sulfoxide (DMSO) and 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for dye (MTT) from MP Biomedicals (France) were used in this study.

The RFP and GFP-expressing U87, U251 and 293 T cells were washed, re-suspended in basic DMEM. For microinjection of tumor cells into the embryos, the concentration of cell is 1 × 10⁷ cells/ml and this mixture was loaded into a borosilicate glass needle pulled by a Flaming/Brown micropipette puller (Narishige, Japan, PN-30). By using an electronically regulated air-pressure microinjector (Harvard Apparatus, NY, PL1–90)^{21 (link)}, 5~10 nanoliters suspension containing about 200–500 cells were injected into the brain of Tg(flk:eGFP) or Tg(flk:mCherry) zebrafish embryos (3dpf), which were anesthetized with 0.04 mg/ml tricaine (MS-222; Sigma, USA). After injection, zebrafish were washed twice with fresh fish water and examined for the presence of fluorescent cells. After each implantation, about 30 fish were selected and cultivated in 6-well plate containing 2 ml of fish water containing with Pen/Strep (1:100) at 33 °C and subsequently documented photographically. Fish water was changed daily, and larva that more than 7 days old were fed twice a day with grinded brine shrimp.