Time lapse images of the third cleaving embryos were acquired on a Zeiss

Time lapse images of the third cleaving embryos were

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enclosure made of plastic tape on a glass slide and sealed with a cover slip. Time-lapse images of the third cleaving embryos were acquired on a Zeiss Axioskop 2 mot microscope equipped with a Zeiss 10× Plan-Neofluar objective lens and a Zeiss Axiocam MRc CCD camera, controlled by Zeiss Axiovision software. The data were obtained every 20 s or 30 s. The third cleavage pattern of each embryo was analysed visually, and the analysed data are summarized in Fig. 2A. Protein detection LsDia1 and β -tubulin protein detection by western blotting was performed as previously described (Kuroda et al., 2016). Fifty one-cell-stage embryos were obtained from one snail for each genotype and used as an SDS-PAGE sample for one lane. The following antibodies were used to probe the respective proteins: anti-LsDia1 (Kuroda et al., 2016; 1/10,000) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Healthcare, NA934; 1/50,000), anti- β -Tubulin (Sigma, TUB2.1; 1/20,000) and HRP- conjugated anti-mouse IgG (GE Healthcare, NA931; 1/50,000). The chemiluminescence signals were imaged using a ChemiDoc MP Imaging System (Bio-Rad) or a Luminescent Image Analyzer LAS-1000plus (FUJIFILM), and the images were analysed using Image Lab Software (Bio-Rad) or Image Gauge (FUJIFILM). Whole-mount in situ hybridization Whole-mount in situ hybridization for the nodal and Pitx genes was performed according to our standard protocols described previously (Kuroda et al., 2009, 2016). Digoxigenin-labelled antisense RNA probes were generated for their full-length coding sequences. Timing of fixing of embryos at the 49- or 64-cell stage was judged by observing the DAPI staining of embryos, whereas that for late trochophore-stage embryos was judged from their appearance. Stained embryo images were acquired using a Zeiss Axio Imager M1 microscope equipped with a Zeiss Axiocam MRc CCD camera. Fluorescent staining Four-cell or four- to eight-cell-stage embryos in the genome-edited snails, dextral (+/+) or sinistral ( - / - ) snails, were fixed in 4% paraformaldehyde in MTSTr [MTS buffer (pH 6.9), 0.1% Triton X-100] at 4°C overnight. Cytoskeleton staining was performed as described previously (Shibazaki et al., 2004; Kuroda et al., 2009, 2016). Confocal images were obtained using a Zeiss LSM 5 pascal laser scanning microscope equipped with a 20× Plan-Neofluar objective lens. The z -stack images were made from optical slices acquired every 10 μ m along the animal-vegetal axis by using Zeiss LSM5 pascal software. One-cell-stage chirality analysis Trypsin treatment for removing the vitelline membrane of the fertilized eggs was performed as described by Meshcheryakov and Beloussov (1975). Crystalline trypsin (Wako) was dissolved in 5×HF before use to prepare a fresh solution at a concentration of 10 mg/ml. Embryos used were obtained from the population of dextral (+/+) or sinistral ( - / - ) and the individual genome-edited snails, 3-F4 (wt/del5) or (del5/del5), which were derived from one self-fertilized heterozygous snail, 3-F1-5-F2-1-F3-1 (wt/del5).
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  • Fall '17
  • Brian Buchwitz

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