Figure 6

Application of OpenEMMU to complex developing organs and tissues

(A) Diagram of an embryonic day 14 (E14) mouse embryo showing the steps involved in isolating the heart and the OpenEMMU protocol.

(B) Flow cytometry results of isolated total heart cells with EdU staining along DNA content (left graph), demonstrating its multiplexing capability with CD31-PE/Cy7 and PDGFRα-PE. The relative proportion of EdU+ cells in both cell populations is depicted (n = 3).

(C) Light-sheet fluorescence imaging of EdU multiplexed with Ki67 and cardiac Troponin T, along with DNA labeling in an E14 heart, shown from a front view (top panels) (n = 4). A top view of the same heart is provided, with EdU labeling color-coded by depth projection. Ra, right atrium; La, left atrium; Pt, pulmonary track; Rv, right ventricle; Lv, left ventricle; Ao, aorta.

(D) Light-sheet imaging of EdU multiplexed with αSMACy3 and p-HH3^S10-A594 immunolabeling, along with DNA staining of an E14 forelimb, shown from dorsal and ventral views (n = 3).

(E) Inset of (D, ventral side of the digit) showing a magnified view of the boxed area using z stack confocal microscopy. The dotted lines indicate the interdigital necrotic zone, where cellular DNA synthesis and mitosis decrease dramatically.