Fig. 2. (A) Separation of GFP-positive visceral endoderm cells from ES-cell aggregates. Afp-GFP ES cells were aggregated for 4 days in the presence of LIF and subjected to FACS. The cells in R2 region were collected as GFP-positive cells and those in R3 as GFP-negative cells. The y axis indicates forwards scatter and the x axis indicates GFP expression (left). Total RNA was isolated from both GFP-positive and GFP-negative cells, and the RNA was subjected to RT-PCR analysis. PCR products were separated on a 2% agarose gel and visualized by ethidium-bromide staining (right). OPN, osteopontin; Ihh, Indian hedgehog; TTR, transthyretin; AFP, -fetoprotein. (B) Oct4 and osteopontin (OPN) expression were downregulated in the outer layer of ES-cell aggregates. After 4 days of aggregation of Afp-GFP ES cells in the presence of LIF, the aggregates were fixed with 3.7% formaldehyde and subjected to frozen section. The sections were incubated initially with anti-Oct4 antibody (1:100 dilution), anti-osteopontin antibody (1:500 dilution) or anti-HNF4 antibody (1:100 dilution), and subsequently with fluorescein-isothiocyanate-conjugated anti-mouse-IgG (1:200 dilution; for Oct4) or anti-goat-IgG (1:200 dilution; for osteopontin and HNF4) (left, red). Endogenous GFP expression derived from Afp-GFP ES cells was detected in the corresponding section (middle, green). Cells were co-stained with 4,6-diamidino-2-phenylindole to demonstrate nuclei (right, blue). Bars, 50 µm.