Anti phh3

For whole-mount heart imaging, the hearts from EC-SCL-Cre-ERT^+/−;R26^mTmG/+ mice at E9.5 (n = 3), E10.5 (n = 3) and E12.5 (n = 4) were micro-dissected and fixed in 4% paraformaldehyde for 5 minutes and placed in cold PBS. Images were captured using a Q Imaging RETIGA EXi camera connected to a Leica DMIRE2 microscope.
Immunofluorescence was performed on 5 μm sections of Bouins fixed and paraffin embedded tissues following standard protocols33 (link)34 (link). Primary antibodies used include anti-GFP (1:500 dilution, Abcam, ab13970), anti-PECAM-1 (1:500 dilution, Santa Cruz, sc1506-R), anti-phh3 (1:200 dilution, Cell Signaling, #9701S), anti-cTnT (1:200 dilution, Abcam, ab8295), anti-Hb (1:500 dilution, DAKO, A0118) and anti-Runx1 (1:500 dilution, Abcam, ab23980). The secondary antibodies used were Alexa Fluor 488 goat anti-chicken IgG (H + L) (GFP), Alexa Fluor 568 donkey anti-rabbit IgG (H + L) (PECAM-1, phh3, Hb and Runx1) and Alexa Fluor 568 donkey anti-mouse IgG (H + L) (1:500 dilution; Life Technologies, Carlsbad, CA). Nuclei were stained with Hoechst 33342 (1:1000dilution, Invitrogen, H3570). Images were obtained using a Leica DFC360 FX digital camera connected to a Leica DFC 480 microscope.

E17.5 NPCs were cultured in monolayer with rh-BMP7 and rh-FGF9 (50 and 100 ng ml⁻¹, R&D systems) in KSFM. Cultures were incubated with 20 μM EdU (Click-iT EdU Alexa-Fluor 488 Imaging Kit, Life Technologies) 4 h after growth factor stimulation and pulse-chased for 20 h. Fixation, permeabilization and Click-iT reaction was performed according to manufacturer's instructions. Cultures were incubated with anti-pHH3 (1:100, Cell Signaling) antibody for 1 h and Alexa-Fluor-568 secondary antibody was used to visualize the staining. Nuclei were stained with Hoechst 33342 (Life Technologies). Images (5–10)were taken per well for each condition with a minimum of three biological replicates from two independent experiments (n=2). Pooled images were analysed by Image-J and number of EdU+ (S-phase) and/or pHH3+ (Mitosis or M-phase) nuclei were counted and divided by the total number of nuclei to determine the percentage of cells in S- and M-phases. Data are represented as percentage of S- or M- phase cells in each condition.