To perform ventricular CM ablation, Tg(vmhc:mCherry-NTR) larvae were treated with 6 mM MTZ (metronidazole, Sigma-Aldrich) in E3 water for 4 h at 3 dpf as previously described [11 (link)]. To modulate signaling pathways, larvae were incubated with the following chemicals for the indicated time period: 100 μM DAPT (Sigma-Aldrich), 12 μM AG1478 (Sigma-Aldrich), 5 μM cardiomogen-1 (Sigma-Aldrich), 7.5 μM dorsomorphin (Sigma-Aldrich), 5 μM LDN193189 (Selleck), or 10 μM rapamycin (Cell Signaling Technology). To stop blood flow, larvae were treated with 1.8 mM tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich) or 10 mM BDM (2,3-butanedione monoxime, Sigma-Aldrich) in E3 water for the indicated time period, and then washed with fresh E3 water.
Tricaine 3 amino benzoic acidethylester
Manufactured by Merck Group
Sourced in United States, Macao
Tricaine (3-amino benzoic acidethylester) is a lab equipment product manufactured by Merck Group. It is a chemical compound used as an anesthetic agent in various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Cardiomogen 1, by Merck Group (2 mentions)
Ldn193189, by Selleck Chemicals (2 mentions)
Tricaine, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Dorsomorphin, by Merck Group (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Ag1478, by Merck Group (1 mentions)
Bdm 2 3 butanedione monoxime, by Merck Group (1 mentions)
K02288, by Selleck Chemicals (1 mentions)
Pd153035, by Selleck Chemicals (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
Axiocam mrm, by Zeiss (1 mentions)
Daspei, by Merck Group (1 mentions)
Smz 18 zoom stereo microscope, by Nikon (1 mentions)
Phenylthiourea ptu, by Merck Group (1 mentions)
13 protocols using tricaine 3 amino benzoic acidethylester
1
Ventricular Cardiomyocyte Ablation in Zebrafish
2
Modulating Zebrafish Cardiovascular Signaling
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To influence blood flow, control or ablated Tg(vmhc:mCherry-NTR) larvae were incubated in E3 water with 1.8 mM Tricaine (3-aminobenzoic acid ethyl ester, Sigma, St. Louis, MO, USA) or 10 mM BDM (2,3-butanedione monoxime, Sigma, St. Louis, MO, USA) directly after ablation for 15 h [43 (link)]. For signaling pathway studies, zebrafish larvae were incubated in E3 water with different drugs or solvent right after ablation for 20 h. A quantity of 100 μM of DAPT (Sigma, St. Louis, MO, USA) was used to inhibit Notch signaling [5 (link)]; 5 μM Cardiomogen-1 (Sigma, St. Louis, MO, USA) was used to inhibit Wnt signaling [51 (link)]; 7.5 μM K02288 (Selleck, Houston, TX, USA) or 5 μM LDN193189 (Selleck, Houston, TX, USA) was used to inhibit Bmp signaling [53 (link),55 (link)]; 1 μM RA (Sigma, St. Louis, MO, USA) was used to activate RA signaling [69 (link)]; 5 μM PD153035 (Selleck, Houston, TX, USA) was used to inhibit EGF signaling [70 (link)].
3
Zebrafish Hair Cell Regeneration
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Pulled-glass capillary micropipettes with tips broken to 10 µm were used to deliver 1 nL of morpholino (MO) into one-celled zebrafish embryos. The MO, including the standard control MO (GeneTools, Philomath, OR, USA), was dissolved in water to the desired concentration (1 mM) and injected into the cytoplasm. The sequence of anti-miR-183 (MO-183) was 5′-CAGTGAATCTACCAGTGCCATA-3′ & MO-182 was 5′-UUUGGCAAUGGUAGAACUCACA-3′. Wild-type zebrafish larvae at 72 hours post fertilization (hpf) were treated with 500 µM neomycin for 2 h. Hair cells from neomycin-treated larvae were stained using a 20-min immersion in 2 mM YO-PRO1 (Invitrogen, Cergy Pontoise, France) 4 h (78 hpf), 12 h (86 hpf), 24 h (98 hpf), and 48 h (122 hpf) following neomycin treatment. The zebrafish larvae were anesthetized with 0.1% tricaine (3-aminobenzoic acid ethyl ester, Sigma, St-Louis, MO, USA) and mo-unted in 3% methylcellulose (Sigma) in a depression slide. The average hair cell number in three posterior neuromasts (P7, P8, and P9) was evaluated under a fluorescent microscope. These posterior neuromasts were selected because they ex-hibit low variability in the number of hair cells, show definite differences in rate of regeneration, and produce clear confocal images due to the thin surrounding tissue. The study design is summarized in Fig. 1 .
4
Fluorescence Imaging of Zebrafish
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Images were taken with a Cell Observer CSU-X1 (Yokogawa) Spinning Disk (Zeiss), AxioCam MRm (Zeiss) and Evolve 512 (Photometrics) or confocal microscope LSM710 (Zeiss). Brightness and contrast were adjusted using Zen blue or gray (Zeiss) and ImageJ. For in vivo imaging of GFP fluorescence, dechorionated zebrafish were incubated with Tricaine (3-amino benzoic acidethylester) (Sigma) for immobilization. Subsequently, zebrafish were mounted in the Metaphor (low melting temperature) agarose (LONZA).
5
Zebrafish Transgenic Lines in Vivo Imaging
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Wild-type AB/TL, and transgenic Tg(mpeg1:eGFPgl22)35 (link), Tg(mpeg1:mCherryFump2)30 (link) and Tg(mpx:GFPi114)29 (link) zebrafish lines, adults and embryos, were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). Breeding of zebrafish adults was approved by the local animal welfare committee (DEC) of the University of the Leiden, under license number 10612 and in compliance with international guidelines specified by the EU Animal Protective Directive 2010/63/EU. All studies in this work was performed on embryos/larvae before the free feeding stage, no adult fish were sacrificed, and experiments did not fall under animal experimentation law according to the EU Animal Protection Directive 2010/63/EU. Adult zebrafish were kept at 28 °C in an aquarium system with light day/night cycle of 14/10 hours, respectively. Embryos were cultured at 28.5 °C in egg water (60 μg/ml sea salt, Seramarin, Heinsberg, Germany). Prior to fungal microinjections or microscopic imaging embryos were anesthetized in egg water medium containing 0.02% (w/v) buffered Tricaine (3-aminobenzoic acid ethyl ester; Sigma-Aldrich, St Louis, MO, USA).
6
Zebrafish Husbandry and Embryonic Development
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Wild type adult zebrafish (AB or TL strain) are maintained in the Aquatic Facility of the Luxembourg Centre for Systems Biomedicine and the Institute of Biology, Leiden University, according to standard protocols [39 ]. Zebrafish eggs were obtained by natural spawning on the day of each experiment, kept in 0.3X Danieau’s solution (14 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 1.5 mM HEPES pH 7.5 and 0.03 M methylene blue) or egg water (60 μg/ml sea salt, Sera Marin, Heinsberg, Germany), and staged by morphology (one-cell stage) for the injections. After each series of injections, the eggs were incubated at 28°C (±0.5) and evaluated up to 5 days post-fertilization (dpf).
Anaesthesia of larvae used for live imaging and COPAS [37 (link),40 (link)] analysis was done with 0.02% buffered Tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich) in egg water.
Anaesthesia of larvae used for live imaging and COPAS [37 (link),40 (link)] analysis was done with 0.02% buffered Tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich) in egg water.
7
Transgenic Zebrafish Cardiac Imaging
8
Zebrafish Care and Sacrifice Protocol
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Wild-type (WT) AB zebrafish were obtained by the European Zebrafish Research Center (EZRC) (Germany). Zebrafish embryos were kept in petri dishes in zebrafish water (1.2 mM NaHCO3, instant ocean 0.1 g/L, 1.4 mM CaSO4, methylene blue 0.00002% w/v) at 28°C until 6 days post fertilization (dpf), then housed in ZebTEC semi-closed recirculation housing systems (Techniplast) at 28°C, pH 7.5 and conductivity 500 µS on a 14/10 light/dark cycle. Adult zebrafish were fed three times a day alternating dry food and brine shrimps. For the experiments, larvae and adult zebrafish were anesthetized using 0.016% w/v tricaine (3-amino benzoic acidethylester, Sigma Aldrich) in zebrafish water and sacrificed by tricaine overdose (0.03% w/v). All the experiments were performed in agreement with EU Directive 2010/63/EU. The experimental protocol was approved by Italian Ministry of Health (Approval Animal Protocol No.1191/2016-PR).
9
Transgenic Zebrafish Cardiac Imaging
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Transgenic zebrafish lines Tg(gata1a:dsRed; cmlc2:gfp) (Fig. 3 , Supplementary Fig. 14 , Video 4 , 9 ), Transgenic zebrafish lines Tg(cmlc2:gfp) (Fig. 3 , Supplementary Fig. 17 , Video 6 ), Tg(fli1:gfp; gata1a:dsRed) (Supplementary Fig. 14 , 20 , Video 7 , 8 ), Tg(myl7:nls-gfp) (Supplementary Fig. 15 , Video 5 ) were used in our experiments. Embryonic fishes were maintained until 3–4 dpf in standard E3 medium, which was supplemented with extra PTU (Sigma Aldrich, MO) to inhibit melanogenesis. Then, the larvae were anesthetized with tricaine (3-amino benzoic acidethylester, Sigma Aldrich, MO) and immobilized in 1% low-melting-point agarose inside FEP (Fluorinated Ethylene Propylene) tube for further imaging. In the imaging of cardiac blood flow, the embryos were injected with gata2a morpholino oligonucleotide at the single-cell stage, to slow down hematopoiesis, and thereby reduce the density of RBCs. All the experiments were performed in compliance with and with the approval of a UCLA IACUC protocol.
10
Neuromast Labeling and Quantification
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One milliliter of neomycin [10 mM stock in dimethyl sulfoxide (DMSO); Thermo Fisher Scientific, BP2669-5] was added to 49 ml sterile E3 egg water to prepare a working solution with a final concentration of 200 µM. Approximately 100 embryos [homozygous mutant and sibling controls (includes +/− and +/+)] at 5 dpf were placed in a 60×15 mm Petri dish. For neuromast staining, embryos were left for 1 h at 28.5°C, followed by three washes and allowed to recover in sterile E3 egg water for 1 h. Embryos with or without neomycin were incubated in E3 blue containing 50 μg/ml 2-[4-(dimethylamino) styryl]-N-ethylpyridinium iodide (DASPEI; Sigma-Aldrich, D0815; specifically to label hair cells) for 30 min, followed by two to three washes. Embryos were anesthetized in 0.4% tricaine (3-aminobenzoic acid ethyl ester; Sigma-Aldrich) and immediately scored by counting the number of visible neuromasts out of total neuromasts on the head and trunk regions of embryos using a Nikon SMZ-18 Zoom Stereo Microscope with GFP filter (further imaging details below).
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