(A) Immunofluorescence staining of E14.5 embryo hearts representative of endocardial cell apoptosis and proliferation using Apoptag (top panels) to mark apoptotic cells (red) or p-HH3 (lower panels) to mark proliferation (red). Additional double-staining for MF20 was used to identify myocardium (green), and Hoechst stain was used to mark the nuclei, and in this case, to delineate the mitral valves. Scale bars: 50 μm. Quantification of endocardial (B) apoptosis and (C) proliferation is demonstrated from n = 4 embryos per group per analysis, with counts equal to or greater than 1000 cells/embryo/defined area. (D) Immunofluorescence staining of myocardial cell apoptosis and proliferation using Apoptag (upper panels) to mark apoptotic cells (red) or p-HH3 (lower panels) to mark proliferation (red) in left ventricle or right ventricle. Additional double-staining for MF20 was used to identify myocardium (green), and Hoechst stain was used to mark the nuclei. Scale bars: 50 μm. p-HH3, phospho-histone H3. Quantification of myocardial (E) apoptosis and (F) proliferation are demonstrated in left ventricle, right ventricle, septum, and total myocardium, with n = 4 embryos per group per analysis, with counts equal to or greater than 1000 cells/embryo/defined area. All data are expressed as mean ± SEM. *P < 0.05; **P < 0.01. P values were derived from 1-way ANOVA with Bonferroni’s post-test when ANOVA was significant.