Figure 5

Carbohydrate expression as determined by lectin binding using immunocytochemistry. Figure 5A–C shows a hESC colony that represents uniform lectin binding. Ricinus Communis agglutinin (RCA) binding in red (5A) is shown throughout this SSEA-4 positive colony in green (5B). The DAPI nuclear stain image (blue) is also shown (5C). Other lectins showed partial binding patterns, such as Vicia Villosa agglutinin (VVA) binding (red), which is shown in a hESC colony (5D) that has uniform SSEA-4 antibody binding (green) (5E). Arrows denote distinct SSEA-4 positive regions lacking VVA binding. DAPI nuclear staining (blue) is also shown (5F). PHA-E binding is shown in two separate images in Figure 5G–H. In 5G there are two adjacent colonies, one that expresses strong binding of SSEA-4 antibody (green) and weak to no binding of PHA-E (red), and an adjacent colony showing binding of PHA-E without SSEA-4 antibody binding. (DAPI nuclear staining in blue). 5H, shows another colony with a streak of stacked cells (as determined by high DAPI expression, see arrow) in the middle of the colony that are beginning to lose SSEA-4 expression (green), but have strong PHA-E binding (red). However, the rest of the colony adjacent to this streak of cells is uniformly positive for SSEA-4 but is lacking PHA-E binding. 5I shows lack of DBA binding and presence of SSEA-4 and DAPI staining. Images and scale bars: 5A-G) 20× magnification, 100 μm. H-I) 10× magnification, 100 μm.