Zebrafish Fluorescent Vesicle Imaging

Zebrafish were maintained at standard conditions (Westerfield, 1993 ). Fertilized eggs of wild type embryos (ABC) were injected at the 1-cell stage with 25 ng/µl of respective plasmid DNA. Plasmid h2afx:EGFP-Rab5c (Clark et al., 2011 (link)) was used to label the Rab5c vesicles. At ∼36 hpf embryos with transient expression of fluorescent markers were chosen and mounted in a 35 mm glass bottom Petri dish (0.17 mm, MatTek), using 0.7% low melting agarose (Sigma) containing 0.08% tricaine and 0.003% PTU. A Leica TCS SP5 confocal microscope with a 40× water objective was used for imaging.

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Brauchle M., Hansen S., Caussinus E., Lenard A., Ochoa-Espinosa A., Scholz O., Sprecher S.G., Plückthun A, & Affolter M. (2014). Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry. Biology Open, 3(12), 1252-1261.

Publication 2014

glass bottom Petri dish low melting agarose TCS SP5 confocal microscope

Agarose Cell Confocal microscope Dish Embryos Fertilized eggs Plasmid Transient Tricaine Zebrafish

Corresponding Organization :

Other organizations : University of Basel, University of Fribourg, University of Zurich

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Variable analysis

independent variables

Injection of 25 ng/µl of respective plasmid DNA into 1-cell stage wild type (ABC) zebrafish embryos

dependent variables

Transient expression of fluorescent markers (h2afx:EGFP-Rab5c) in ∼36 hpf embryos

control variables

Maintenance of zebrafish at standard conditions (Westerfield, 1993)
Mounting of embryos in 0.7% low melting agarose (Sigma) containing 0.08% tricaine and 0.003% PTU
Imaging with a Leica TCS SP5 confocal microscope with a 40× water objective

controls

No positive or negative controls were explicitly mentioned in the provided information.

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