Zebrafish embryos were injected at the one-cell stage with a glass microneedle and a microinjector (World Precision Instruments). For transgenesis tol2 mRNA, transcribed from pCS2FA with the mMESSAGE mMACHINE SP6 Transcription Kit, a kind gift from Koichi Kawakami, was injected at a concentration of 25 ng µl−1 together with Tol2 destination vectors69 (link). For mutagenesis Cas9 mRNA was in vitro transcribed from the MLM3613 plasmid (Addgene plasmid #42251) using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific); sgRNA target sequences were cloned into DR274 (Addgene plasmid # 42250) and transcribed with the MAXIscript T7 transcription kit (Thermo Fisher Scientific). Subsequently, 1 nl of a mixture of 600 ng ml−1Cas9 mRNA and 50 ng ml−1 sgRNA were co-injected into one-cell stage embryos29 (link).
Glass microneedle
Manufactured by World Precision Instruments
The Glass Microneedle is a precise and delicate laboratory tool designed for a variety of applications. It features a sharp, tapered tip made of glass, enabling the user to manipulate and interact with small-scale samples and materials with exceptional control and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Microinjector, by World Precision Instruments (3 mentions)
Maxiscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions)
Texas red dextran, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Glass bottom microscopy dishes, by MatTek (1 mentions)
Nusieve gtg agarose, by Lonza (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
4 protocols using glass microneedle
1
Zebrafish Transgenesis and Mutagenesis
2
Microangiography of Zebrafish Embryos
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Embryos were anaesthetized with 0.112 mg ml−1 Tricaine in E3 medium and mounted in 0.7% (w/v) low-melting agarose (NuSieve GTG Agarose, Lonza) in glass bottom microscopy dishes (MatTek). For the microangiography Texas Red-dextran with a molecular weight of 70 kDa (Thermo Fisher Scientific, Supplementary Table 6 ) was solubilized in E3 medium to a concentration of 2 mg ml−1. Using a glass microneedle and a microinjector (World Precision Instruments), the dextran was injected into the sinus venosus. Subsequently, images were taken with an SP8 confocal microscope (Leica).
3
Evaluating Vascular Permeability in Zebrafish
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The experiments were performed as previously described with minor modifications (Hoeppner et al., 2012 (link)). Capped Cre mRNA was synthesized using mMessge mMachine T3 transcription kit (Thermo Fisher Scientific) and microinjected into embryos at 1–2 cell stage (1.5 nl, 12.5ng/ul). At 3-days post-fertilization (3-dpf), embryos were anaesthetized in 0.015% tricaine methanesulfonate (Western Chemical, Inc) and microangiography was performed by inserting a glass microneedle (World precision Instruments, Sarasota, FL) through the pericardium directly into ventricle as described previously (Hoeppner et al., 2012 (link)). Texas Red-dextran with a molecular weight of 70 kDa solubilized in embryo medium at a 2 mg/mL concentration and a total of 4.5 nL was injected. Basal vascular permeability in zebrafish was defined as no exposure to heat shock. Acute vascular permeability was induced by 1 heat-shock induction, which was performed by transferring the zebrafish from 28.5°C to 37°C embryo water and incubating at 37°C for 30 minutes. Chronic vascular permeability was defined as three 30-minute heat-shock inductions of VEGF separated by 30 minutes at 28.5°C. Images were acquired immediately after heat-shock using a Zeiss LSM 880 confocal microscope using standard FITC and dsRed filter sets and 10X objective at room temperature.
4
Transgenesis and Mutagenesis in Zebrafish
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Zebrafish embryos were injected at the one-cell stage with a glass microneedle and a microinjector (World Precision Instruments). For transgenesis Tol2 mRNA, transcribed from pCS2FA (a kind gift from Koichi Kawakami) with the mMESSAGE mMACHINE SP6 Transcription Kit was injected at a concentration of 25 ng µl−1 together with Tol2 destination vectors69 (link). For mutagenesis, 1 nl of a mixture of Cas9 protein (0.5 µg/uL, IDT) and sgRNA (3 µM, IDT) were co-injected into one-cell stage embryos. The sgRNAs that have been used to create the esm1 mutant (formally named esm1ka616) are indicated in Suppl. Table 1 . The primers used to genotype the esm1 mutant are indicated in Suppl.Table 2 .
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