Anti phosphorylated histone 3 antibody

Plasmid-containing junb-l (PCRII-junb-l), was kindly sent by Atsushi Kawakami (Department of Biological Information, Tokyo Institute of Technology). Digoxigenin (DIG)-labeled (Roche, France) sense and antisense RNA probes were obtained by in vitro Transcription (Biolabs, France). In situ hybridizations on whole-mount embryos were performed as previously described.^{46 (link)} Stained embryos were imaged using a MVX10 Olympus microscope (MVPLAPO × 1 objective and XC50 camera) and using a Zeiss Axioimager (Zeiss × 40 Plan-Apo 1.3 oil objective) (Zeiss, France). For quantification of cell proliferation, whole embryos were fixed in paraformaldehyde 4% and stained as described in ref. 46 (link) using an anti-phosphorylated histone 3 antibody (Cell Signaling, France, ref 9701, 1/500) and a secondary antibody Goat anti-Rabbit coupled with Alexa Fluor 488 (A-11034, Life Technologies-Invitrogen, France). For cell death quantification, the embryos were placed in Petri dishes containing 5 μg/ml of Acridine orange (Sigma-Aldrich, France) diluted in zebrafish water from a 10 mg/ml stock solution and incubated during 30 min. Embryos were then washed with zebrafish water three times 10 min each. The embryos were then replaced at 28 °C until observation by confocal microscopy.

For quantification of cell proliferation, whole embryos were fixed in 4% paraformaldehyde overnight and stained using an anti-phosphorylated histone 3 antibody (Cell Signaling, ref 9701, dilution:1/500)^{12 (link)}.