Fig. 6. Cell proliferation study of AV cushions. (A-H) Frontal sections of control (A,B,E,F) and mutant (C,D,G,H) embryonic hearts at E11.5 stained with DAPI to visualize all nuclei (A,C,E,G) and with anti-Ki67 antibody to identify proliferating cells (B,D,F,H). A-D and E-H show the inferior and superior AV cushions, respectively. (I) The number of Ki67-positive nuclei as a percentage of total nuclei. Data (mean±s.d.) were collected from four embryonic hearts of each strain, and at least 500 nuclei were counted for each heart. (J-O) Frontal sections of control (J-L) and mutant (M-O) embryonic hearts (E11.5) stained with propidiumiodide (J,M) and an anti-cyclin D1 antibody (K,N). Only results from inferior cushions are shown. Identical confocal microscopic conditions were used to acquire the images of control and mutant samples. (L) Merged images of control (J,K) samples. (O) Merged images of mutant (M,N) samples. Arrows in M-O indicate examples of nuclei without cyclin D1. (P) Relative intensity of cyclin D1 immunostaining in mutant versus control hearts. The total intensity of cyclin D1 antibody staining (green) was determined using MetaMorph 5.0, and was averaged with the number of nuclei to acquire the average intensity. The intensity of the control superior cushion was set as 100%. (Q) The number of cyclin D1-positive nuclei as a percentage of total nuclei. For P and Q, data (mean±s.d.) were collected from five embryonic hearts of each strain, and at least 500 nuclei were counted for each heart. ^*P<0.05. cko, Tie2cre;Tgfbr2^loxp/loxp;R26R; ctrl, Tie2cre;Tgfbr2^loxp/+;R26R; IC, inferior cushion; SC, superior cushion; PI, propidiumiodide.