mRNA encoding Lefty1- or Lefty2-GFP33 (link) was generated by plasmid linearisation with NotI (NEB), purification with a Qiagen PCR clean-up kit, and in vitro transcription using SP6 mMessage mMachine kits (Ambion). Pronase-dechorionated wild type (TLAB) embryos at the one-cell stage were injected with different amounts of lefty1-GFP (22 pg, 43 pg, 86 pg) or lefty2-GFP (5 pg, 10 pg, 20 pg) mRNA along with 100 pg of 10 kDa Alexa546-dextran (Life Technologies). At sphere stage, three to five embryos per condition were imaged on an LSM 780 (ZEISS) confocal laser scanning microscope, and eight embryos with three replicates per condition were collected for quantitative reverse transcription PCR (qRT-PCR) at 50% epiboly. Extracellular fluorescence intensity quantifications and qRT-PCR measurements with Promega Go-Taq qPCR Master Mix were executed as described previously33 (link) using the zebrafish elongation factor eF1α as a normalisation control.
Alexa546 dextran
Manufactured by Thermo Fisher Scientific
Alexa546-dextran is a fluorescent conjugate of the dye Alexa Fluor 546 and dextran. It is used as a tool for biological research, providing a fluorescent marker for visualizing and tracking dextran-containing structures or processes within cells and tissues.
Automatically generated - may contain errors
2 protocols using alexa546 dextran
1
Lefty1 and Lefty2 mRNA Injection in Zebrafish
2
Lefty1 and Lefty2 mRNA Injection in Zebrafish
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mRNA encoding Lefty1- or Lefty2-GFP33 (link) was generated by plasmid linearisation with NotI (NEB), purification with a Qiagen PCR clean-up kit, and in vitro transcription using SP6 mMessage mMachine kits (Ambion). Pronase-dechorionated wild type (TLAB) embryos at the one-cell stage were injected with different amounts of lefty1-GFP (22 pg, 43 pg, 86 pg) or lefty2-GFP (5 pg, 10 pg, 20 pg) mRNA along with 100 pg of 10 kDa Alexa546-dextran (Life Technologies). At sphere stage, three to five embryos per condition were imaged on an LSM 780 (ZEISS) confocal laser scanning microscope, and eight embryos with three replicates per condition were collected for quantitative reverse transcription PCR (qRT-PCR) at 50% epiboly. Extracellular fluorescence intensity quantifications and qRT-PCR measurements with Promega Go-Taq qPCR Master Mix were executed as described previously33 (link) using the zebrafish elongation factor eF1α as a normalisation control.
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