Fig. 6. TGF-induced acinar-to-ductal metaplasia proceeds through nestin-positive intermediates. Images from day 0 (A,D,G,J), day 2 (B,E,H,K) and day 5 (C,F,I,L) TGF-treated non-transgenic pancreatic explants, double immunolabeled for: (A-C) nestin (green) and amylase (red); (D-F) nestin (green) and ductal cytokeratins (red). Owing to high cytokeratin and low nestin expression, composite color is orange rather than yellow (arrowheads); unmerged images are provided in Fig. S2 in the supplementary material. (G-I) Ductal cytokeratins (green) and amylase (red); (J-L) carbonic anhydrase II (green) and amylase (red). Nuclei are labeled in blue. On day 0, there were no nestin-positive cells and no cells co-expressing acinar and ductal markers. Following only 2 days of culture, cells had reduced levels of amylase and were beginning to acquire ductal cytokeratins. Following 5 days of culture, amylase protein was no longer detected, and cells had acquired expression of both ductal cytokeratins and carbonic anhydrase II. All images are presented at identical magnifications.