Fig. 3. Differentiation of EPL cell aggregates in MEDII results in the formation of homogeneous populations of neurectoderm. (A) 15 µg of RNA isolated from EBM^6-9 was analysed for the expression of Sox1 and mGAP by RNase protection. (B) Immunohistochemical analysis for the presence of the neurofilament protein nestin in a seeded EBM⁷ aggregate after a further 48 hours culture. Size bar: 210 µm. (C,D) EB7 and EBM7 were seeded into individual 2 ml wells and examined on days 8, 10 and 12 for the formation of beating cardiocytes (C) and neural extensions (D). n>48/experiment, 5 experimental repeats represented. (E,F) EBM⁷ were seeded and cultured for a further 4 days in serum-free medium before analysis for the presence of NeuN (E) and tubulin-ß III (F). (G,H) EBM⁹ were analysed by whole-mount in situ hybridisation for the expression of Sox1 (G) and Sox2 (H) using digoxigenin-labelled anti-sense probes. After colour development, aggregates were fixed, embedded and cut into 7 µm sections. Sections were viewed under brightfield microscopy. Size bar: 210 µm. (I) EBM¹⁰ were disaggregated, probed for the expression of NCAM by immunohistochemistry and analysed by flow cytometry. The bar, which indicates positive fluorescence, was determined experimentally by analysis of cells probed with secondary antibody alone (data not shown).