Phospho histone h3 ser10 antibody

For the immunofluorescence assay, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. After washing, the cells were permeabilized with 0.1% Triton X for 10 min at room temperature. The cells were then incubated with phospho-histone H3 (Ser10) antibody (1:100 dilution; Cell Signaling Technology) at 4 °C overnight. Then, the cells were incubated with Alexa Fluor 568-conjugated secondary antibody (1:100 dilution, Invitrogen, USA) for 1 h at 37 °C. After washing the cells with PBS, nuclei staining was performed with DAPI for 10 min at room temperature. Images were captured using a fluorescence microscope (Olympus).

We performed whole-mount immunohistochemistry using an anti-MF20 (Developmental Studies of Hybridoma Bank) antibody, anti-MEF2 (sc-313; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody to count cardiomyocytes at various stages of development, and Phospho-Histone H3 (Ser10) Antibody  (9701, Cell Signaling Technology, Danvers, MA) to count proliferating cardiomyocytes. The embryos were grown to the desired developmental stage and subsequently fixed overnight at 4 °C in 4% PFA and washed with PBS containing 0.1% Tween 20 the following day. Immunofluorescence was performed as described previously^{88 (link)}. The embryos were flat-mounted in 50% glycerol and imaged ventrally using a Carl Zeiss confocal microscope LSM 510 META, with a 20-μm lens for the heart tube overview and a 60-μm immersion lens for cell count of the ventricle and atrium. Sequential confocal images were taken with excitation at 488 and 568 nm, and a standardised z-stack size of 0.642 μm. Three-dimensional (3D) reconstructions of confocal stacks were made using 3D projection software (Carl Zeiss). The MEF2- and MF20-positive cells were confirmed on each z-stack. All embryos were counted at least three times.

Cells were maintained in culture with no treatment or stimulated with 0.5 μM taxol (Sigma) for 20 h. Harvested cells were fixed and permeabilized in 70% ethanol at 4°C for 2 h. After a washing step, cells were resuspended in PBS+1% BSA at 10⁶ cells ml⁻¹. Cells were incubated with phospho-histone H3 (Ser10) antibody (Cell Signaling) at 1:70 dilution for 20 min, washed and subsequently incubated with Alexa Fluor 488 fluorochrome-coupled secondary antibody (Invitrogen) at 20 μg ml⁻¹ for 20 min. After washing with PBS+1% BSA, cells were incubated with 7AAD (BD Biosciences) for 10 min. Cells were acquired in a FACSCalibur cytometer (BD Biosciences) and data were analysed using FlowJo software v9.3.

In total, 16, 18 and 20-day old opossums were cryosectioned, and put through immunofluorescence (IF) staining to highlight apoptosis (using EMD Millipore ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110), an indirect TUNEL method), autophagy (using Anit-LC3B antibody (ab51520 from Abcam), and cellular proliferation (using Phosphohistone H3 (Ser10) antibody from Cell Signaling Technology) [25 (link),26 (link)]. More detailed IF methods can be found in the electronic supplementary Materials and Methods.

For annexin-V and Phospho-Histone H3 (pSer^{12 (link)}) detection, the forelimb bud of embryos at time 0 or cultured for 1, 3 and 6 hours were harvested and incubated for 15 minutes in trypsin, 5% EDTA (Invitrogen). Then, tissues were disrupted mechanically and the released cells washed in complete media. For detection of annexin-V detection, cells were washed with binding buffer and incubated with annexin-V FITC (BD Pharmingen, Le Pont-de-Claix, France) during 15 minutes at room temperature. For Phospho-Histone H3 detection, cells were permeabilized with the intranuclear detection Perm/Fix solution (eBioscience) and stained 1 hour with the Phospho-Histone H3 (Ser10) antibody (Cell Signaling Technology). Then, cells were washed and stained 30 minutes with anti-rabbit Alexa 647 antibody (Cell Signaling Technology). Finally, samples were acquired on the FACS Canto II and analyzed using the FlowJo software (Ashland, OR, USA).

Cell proliferation was monitored by immunohistochemistry. Paraffin sections of E13.5-14.5 outflow tract septal cushions from Tgfb3^−/− and wildtype embryos were subjected to immunohistochemistry with a phospho-histone H3 (Ser10) antibody (Cell Signaling Inc, #9701, Danvers, MA, USA). Stained samples were observed with a Nikon E400 light microscope and photographs (×Obj) were used for quantitative analysis (Image Pro Plus software) by analyzing the number of pHH3-positive cells per sample collected from the region around fibrous OFT septum in 4–5 sections spaced 24 µm apart for 3 samples each of Tgfb3^−/− and control embryos.