Isolation and live imaging of kidneys from E12.5 embryos. (A-C) Separation of embryo proper from placenta and yolk sac. (E) Embryo; (P) placenta; (Y) yolk sac; (arrow) location of umbilical cord. (D) Removal of the head aids dissection and is a convenient source of tissue for genotyping. (E-H) Isolation of urogenital system. (A) Anterior; (P) posterior. The embryo is pinned with forceps at the level of the heart, and superficial organs are removed so that the urogenital system (F, dashed lines) is visible. Arrows indicate anterior end of nephric ducts, where “peeling” (G, curved arrow) is initiated. Kidneys will appear at the posterior end of the urogenital system, at the level of the hindlimb (H, arrows). (I,J) Kidneys (arrows in I) are separated from the urogenital system. (K,O) Live imaging of isolated kidneys at the beginning of the organ culture (t = 0; K,L) and after 2 d of culture (t = 2 d; M-O). Brightfield images (K,M,N) demonstrate growth of cultures. Developmental structures such as the metanephric mesenchyme (blue dotted line), ureteric bud (red dotted line), renal vesicle (black arrow), and S-shaped body (white arrow) are easily identified at higher magnifications (N). Live GFP from the Hes1-GFP transgene (L,O) labels the S-shaped body of the forming nephrons.