Fig. 1. Alcian-blue-stained areas loose their stainability during differentiation of ES cells in vitro. Alcian blue staining demonstrating differentiation of chondrocyte nodules in ES-cell-derived EBs at 5+21 d (A), 5+26 d (B) and 5+31 d (C). The size of the stained areas was measured during EB cultivation from 5+7 d up to 5+31 d (D). Eleven days after plating (5+11 d) the first stained areas were detected, their number and size increased during cultivation up to 5+18 d and decreased during later stages. Mean values±s.e.m. are shown (n=10). The decrease in the size of Alcian-blue-stained areas was caused by a loss of stainability as demonstrated for nodules from 5+21 d, 5+26 d and 5+31 d (A-C) EBs. Representative areas of EB outgrowths are shown. Bar, 100 µm.