Whole-mount antibody labelling was carried out using standard protocols [47 ]. Briefly, zebrafish were terminally anaesthetised in tricaine (3-amino benzoic acid ethyl ester, Sigma) and incubated immediately in primary fixative (2% paraformaldehyde/ 1% trichloroacetic acid, or 4% paraformaldehyde). Samples were then permeabilized in acetone at -20°C or 10pg/ml proteinase K at room temperature, blocked in 10% normal goat serum and incubated with primary antibodies (3A10 antibody at 1:200 dilution, Developmental Studies Hybridoma Bank; HB9 antibody MNR2 at 1:400 dilution, Developmental Studies Hybridoma Bank; phosphorilated-histone 3 Ser10 antibody at 1:200 dilution, Cell Signalling #9701; all antibodies incubated with 5% normal goat serum). After washing, samples were incubated with appropriate secondary antibodies conjugated with AlexaFluor Dyes Al488, Al568 or Al633 (1:2000 dilution; Molecular Probes, Life Technologies).
3 aminobenzoic acid ethyl ester
Manufactured by Merck Group
Sourced in United States
3-aminobenzoic acid ethyl ester is a chemical compound. It is a colorless crystalline solid.
Automatically generated - may contain errors
Lab products found in correlation
1 phenyl 2 thiourea, by Merck Group (3 mentions)
Ms 222, by Merck Group (2 mentions)
3a10 antibody, by Developmental Studies Hybridoma Bank (1 mentions)
Axio examiner d1, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Tricaine, by Merck Group (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Lidocaine, by Bio-Techne (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Glycine, by Merck Group (1 mentions)
Collagenase b, by Roche (1 mentions)
Nanoliter 2000, by World Precision Instruments (1 mentions)
Phenylthiourea ptu, by Merck Group (1 mentions)
Astaxanthin, by Merck Group (1 mentions)
20 protocols using 3 aminobenzoic acid ethyl ester
1
Whole-mount Antibody Labeling of Zebrafish
2
Incision and Transection of Dorsolateral Fasciculus
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Larvae were anesthetized in 0.02% 3-aminobenzoic acid ethylester (Sigma-Aldrich Co. LLC.) and embedded in 2% LMP agarose. The neural tube was incised at the second-somite level using a razor blade under stereomicroscopy, and its complete disjunction was confirmed at the end of experiment following fixation in 4% paraformaldehyde (PFA; Nakalai Tesque, Inc.). Transection of the DLF was performed under an upright microscopy (Axio Examiner D1, Carl Zeiss GmbH) using a sharp glass capillary attached to a micromanipulator (Leica Microsystems GmbH). The DLF was labeled with GFP to guide the accurate incision and minimize artificial destruction of surrounding tissues.
3
Zebrafish Husbandry and Experiments
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Zebrafish husbandry and experiments were conducted in compliance with guidelines from the Zebrafish Model Organism Database (http://zfin.org ), the EU Animal Protection Directive 2010/63/EU, and the directives of the local animal welfare committee of Leiden University (License number: 10612). All wild‐type (WT), mutant, and transgenic lines used in this study were generated in the AB/TL background. The zebrafish lines used were: WT‐AB/TL, homozygous mutant (cxcr3.2–/–) and WT siblings (cxcr3.2+/+) of cxcr3.2hu6044, homozygous mutant (cxcr3.3–/–) and WT siblings (cxcr3.3+/+) of cxcr3.3ibl50, and the same lines crossed into Tg(mpeg1: mCherry‐F)ump2 background and Tg (mpx: eGFP)i114,36 and homozygous mutants (dram1–/–) and wild type siblings (dram1+/+) of dram1ibl53.37 Eggs and larvae were kept at 28.5°C in egg water (60 μg/ml Instant Ocean sea salts and 0.0025% methylene blue). All larvae were anesthetized with 0.02% buffered tricaine, (3‐aminobenzoic acid ethyl ester; Sigma‐Aldrich, St. Louis, MO, USA) before infection, tail‐amputation, and imaging. Larvae were kept in egg water containing 0.003% PTU (1‐phenyl‐2‐thiourea; Sigma‐Aldrich) to prevent pigmentation before confocal imaging.
4
Zebrafish Lines Handling and Imaging
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Zebrafish lines were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). The study was approved by the local animal welfare committee (DEC) of the University of Leiden (licence number: 10612, protocol 14227). Fish lines used in this work were the following: wildtype strain AB/TL, Tg(il1b:eGFP-Fzf550)38 (link), Tg(fli1a:eGFPy1)49 (link), the double transgenic line Tg(mpeg1:mCherry-Fump2/mpx:eGFPi114)50 (link)51 (link), cxcr4b homozygote mutant (cxcr4b−/−) and wildtype (cxcr4b+/+) siblings of (cxcr4bt26035)12 (link)17 (link) crossed into the transgenic backgrounds of Tg(kdrl:eGFPs843)29 (link) or Tg(mpeg1:mCherry-Fump2). Embryos were grown at 28.5 °C in egg water (60 μg/ml sea salt, Sera Marin, Heinsberg, Germany). Larvae destined to image acquisition were maintained in egg water supplemented with 0.003% PTU (1-phenyl-2-thiourea, Sigma-Aldrich, St Louis, MO, USA) from 8–12 hpf to prevent melanisation. Anaesthesia of embryos/larvae used for live imaging was achieved with 0.02% buffered Tricaine (3-aminobenzoic acid ethyl ester, Sigma-Aldrich) in egg water.
5
Zebrafish Model for Collagen Disorder
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Wild-type AB (WT) and heterozygous Chihuahua (col1a1adc124/+, Chi/+) zebrafish arise from natural spawning or from in vitro fertilization. The mutant Chi/+, provided by Professor Shannon Fisher (Dept of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, USA), carries a G2207A mutation in col1a1a, causing a p.G736D (G574D) substitution in the α1 chain of type I collagen. Embryos were kept in Petri dishes in fish water (NaHCO3 1.2 mM, instant ocean 0.1 g/L, CaSO4 1.4 mM, methylene blue 0.00002% w/v) at 28 °C until 6 day post-fertilization (dpf), then housed in ZebTEC semi-closed recirculation housing systems (Tecniplast) and kept at a constant temperature (27–28 °C), pH (7.5) and conductivity (500 μS) on a 14/10 light/dark cycle. For the experiments stated below larvae and adult fish were anesthetized using a solution of tricaine (3-amino benzoic acidethylester, Sigma Aldrich) 0.0016% w/v, in fish water and sacrificed by tricaine overdose (0.03% w/v). All experiments were performed in accordance with the approved guidelines, in agreement with EU Directive 2010/63/EU for animals. The experimental protocol was approved by the Italian Ministry of Health (Approval Animal Protocol No. 1/2013).
6
Xenopus Oocyte Isolation and Maintenance
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Female Xenopus laevis frogs were anaesthetised by submersion for 30 min in 3-Aminobenzoic acid ethyl ester (1.5 g/L) (Sigma-Aldrich, St Louis, MO, United States). Anesthetized frogs were placed on ice to slow blood flow and surgery was conducted as previously described (Broer, 2010 (link); Parker et al., 2019 (link)). Pieces of ovary were transferred into buffer (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM Hepes-NaOH, pH 7.8) in a fresh Petri dish and cut into small clumps of 15 to 30 oocytes and were digested in 1.5 mg/ml collagenase D and 0.1 mg/ml collagenase A (Sigma-Aldrich) dissolved in (pH 7.8) buffer for 2 4 h at 28°C. Following digestion, oocytes were washed and examined for the extent of defolliculation and individual separation. Defolliculated oocytes were maintained in OR2+ buffer (OR2− supplemented with 1.5 mM CaCl2 and 50 μg/ml gentamycin, pH 7.8) at 16 18°C. Maintenance of animals and preparation of oocytes was approved by the Australian National University animal ethics review board (ANU Protocol A2017/36).
7
Zebrafish Embryo Handling and Anesthesia
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The work was carried out at the Hubrecht Institute (the NL), according to local laws. Ethical approval was obtained through the relevant DEC committee (HI 10.1801). Standard husbandry conditions applied, according to FELASA guidelines [47 (link)]. Embryos were kept in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) at 28°C. For anesthesia, a 0.2 % solution of 3-aminobenzoic acid ethyl ester (Sigma), containing Tris buffer, pH 7, was used.
8
Zebrafish Husbandry and Pigmentation Inhibition
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Wildtype (AB strain) and Sirt1-/- zebrafish were raised in a standardized aquaria system (Genomic-Design Co., Daejeon, Korea) (http://zebrafish.co.kr ). The system provided continuous water flow, biofiltration tank, at a constant temperature of 28.5°C, UV sterilization, and 14/10 h light/dark cycle. Embryos to be processed for whole-mount analyses were placed in an E3 medium with 0.003% phenylthiourea at 24 h after fertilization to inhibit pigmentation. Embryos and adult zebrafish were anaesthetized in 0.02% tricaine (3-aminobenzoic acid ethyl ester, Sigma) before sacrifice for analysis. This study was approved by Institutional Animal Care and Use Committee of Yonsei University Health System (IACUC of YUSH: 2019–0036).
9
Xenopus Oocyte Glycine Receptor Assay
10
Comparison of Energy Reserve Management in Male Fish
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To compare the energy reserve management among different male types, we also haphazardly collected 10 immature individuals, 10 potential sneaker males, and three dwarf males at Wonzye Point, plus five additional dwarf males at Kasakalawe, a location ∼7.8 km from Wonzy Point. Immature males are usually roaming about in groups to search for food, whereas sneaker males either roam about in groups as well, or stay in the proximity of a nest male's territory where they may attempt to enter a nest to steal fertilizations. Dwarf males are generally harder to find because they cannot be identified unequivocally by their body size and morphology. Only their behavior when trying to enter a nest provides clear information about their tactic. We validated the assignment of all collected males to different tactics by the states of their testes. For collection, all males were first anaesthetized and finally killed with an overdose of MS222 (3-aminobenzoic acid ethyl ester, Sigma-Aldrich Chemie GmbH, Buchs SG, Switzerland). The standard length (SL to the nearest 0, 1 mm) and body mass (BM, 0.1 mg accuracy) of all fish was measured shortly after collection.
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