Analysis of apoptotic cell death in ARF6−/− embryonic liver. (A and B) E13.5 ARF6−/− embryonic liver sections were double stained with TUNEL (green) and antialbumin (to identify hepatocytes [red]) (A) or anti-Ter119 antibodies (to identify erythroid cells [red]) (B). TUNEL-positive and -negative cells are indicated by arrowheads and arrows, respectively. (C and D) Liver sections prepared from E10.0 wild-type (Wt) (C) and ARF6−/− (D) embryos were double stained with TUNEL (green) and PI (red). Arrowheads represent TUNEL-positive liver cells, and dotted lines show the outlines of the livers. (E) In vitro-cultured fetal hepatocytes prepared from E13.5 embryonic livers were stained with TUNEL. TUNEL-positive cells were counted to calculate the percentages of apoptotic cells (n = 3). Data shown are the means ± SDs. (F) Hematopoietic cells prepared from E13.5 embryonic livers were cultured for 3 days. To evaluate the extent of apoptosis, the harvested cells were stained with annexin V-FITC and the percentages of annexin V-positive cells in PI-negative cells assessed by flow cytometry (n = 3). Data shown are the means ± SDs. G, gut; L, liver; scale bars, 10 μm (A and B) and 50 μm (C and D).