Figure 3: Double labeling for the epithelial marker cytokeratin and the myofibroblast marker SMA was used to determine if cell outgrowths were undergoing EMT. Two-colour immunofluorescence was performed at 3, 10, and 17 days after explanting. By labelling for both SMA (red) and cytokeratin (green), outgrowths were initially epithelial at day 3. By day 10 cell outgrowths expressed both SMA and cytokeratin (yellow) indicating that cells were coexpressing epithelial and mesenchymal markers. By day 17, cells had developed a mesenchymal phenotype, and all stained positive for SMA. Scale bar = 25 μm.