For whole-mount heart imaging, the hearts from EC-SCL-Cre-ERT+/−;R26mTmG/+ 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.
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.
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