Gtx128116

Cell proliferation and G₂ phase length were analyzed by specific experimental design described previously (Fig. 6a). Embryos were first treated with 10 mM 5-bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich) pulses as described62 at 16 hpf and then fixed with fresh 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) every hour until 19 hpf. Double IHC was performed with mouse BrdU antibody (B2531, Sigma-Aldrich, 1:250) and rabbit P-H3 antibody (GTX128116, Genetex, 1:1000) to mark cells in S phase and in M phase with corresponding secondary antibodies (1:500). Images were collected utilizing LSM 780 confocal laser-scanning microscope (Carl Zeiss) with 20X lens scanning the dorsal midbrain. Every individual embryo was scanned reaching 25 μm deep from top (10 stacks, 2.5 μm per stack) and shown with represented z stack. Furthermore, G₂ phase length was determined by the percentage of labeled mitosis (PLM) paradigm28 (link) which reveal the progression of the BrdU and P-H3 co-labeled nuclei after BrdU incorporation. Due to its proportionality to G₂ length, it is simple to predict the duration of G₂ phase by 50% of co-labeled nuclei in average.

Whole-mount immunohistochemistry (IHC) staining was performed as previously described (Inoue and Wittbrodt, 2011 (link)) by using either mouse anti-GFP antibody (GT859, GeneTex, 1:500), rabbit anti-histone H3S10ph (phosphor Ser10) antibody (GTX128116, GeneTex, 1:1000), mouse anti-ZO-1/TJP1 antibody (33-9100, Thermo Scientific, 1:100), rabbit anti-GFP antibody (GTX113617, GeneTex, 1:500), mouse anti-tubulin (acetyl Lys40) antibody (32-2700, Thermo Scientific, 1:1000), and rat anti-mCherry antibody (M11217, Molecular probes, Thermo Scientific, 1:300). Secondary antibodies used are goat anti-mouse or anti-rabbit IgG conjugated with Alexa Flour 488 or Alexa Fluor 568 (Molecular probes, 1:500). Confocal images were collected utilizing LSM 780 or LSM 880 confocal laser-scanning microscope with a 20X lens or 43X water lens (Carl Zeiss, Oberkochen, Germany). In general, 8 to 20 layers with 1-μm thickness were scanned and stacked for each image unless otherwise stated, further processed, and presented as maximum intensity projection by the Fiji software (Schindelin et al., 2012 (link)).