Images for phenotypical analyses were taken with the AxioCam MRc 5 microscope (Zeiss). Embryos were scored blind and data are representative images from 2 or more independent experiments. Representative embryos were selected using Magnetic Lasso tool of Adobe Photoshop CS6. Background was adjusted uniformly for presentation. Phenotypical features were analyzed by comparison to control embryos and classified and ‘normal’ and ‘defect’ (showing DV/AP patterning defects).
Quantifying Ciliogenesis Defects in Xenopus Embryos
Images for phenotypical analyses were taken with the AxioCam MRc 5 microscope (Zeiss). Embryos were scored blind and data are representative images from 2 or more independent experiments. Representative embryos were selected using Magnetic Lasso tool of Adobe Photoshop CS6. Background was adjusted uniformly for presentation. Phenotypical features were analyzed by comparison to control embryos and classified and ‘normal’ and ‘defect’ (showing DV/AP patterning defects).
Corresponding Organization : Institute of Molecular Biology
Other organizations : DKFZ-ZMBH Alliance, Heidelberg University, University Hospital Münster
Variable analysis
- Knockdown/overexpression of genes/proteins (e.g., LRP6 WT, LRP6^VA, LRP6^(VA)P(PA))
- Injection of ciliary GSK3 biosensor
- Ciliogenesis defects in X. tropicalis MCC cells (classified as 'Normal', 'Defect' (lower cilia density and shorter cilia), 'Mild' (>half the length compared to control), 'Severe' (
- Ratio of MCC or Ionocytes to total cells in X. tropicalis embryos
- PLrp6 staining intensity normalized to AcTub+ area for each MCC
- LRP6 WT, LRP6^VA and LRP6^(VA)P(PA) staining intensity normalized to AcTub+ area for each MCC
- Mean signal intensity of ciliary GSK3 biosensor normalized to first time point (t=0) or to Arl13b-mKate2 mean intensity
- Imaging conditions (Zeiss LSM 700 microscope, Nikon AX confocal microscope)
- Imaging parameters (pinhole set to 1 Airy unit, pixel size, z-sections)
- Image analysis software (ImageJ/Fiji)
- Staining/labeling methods (acetylated tubulin, phalloidin, Arl13b-mKate2)
- Control embryos for phenotypical analysis
- No mention of positive or negative controls
Annotations
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