Fig. 2. Immunohistochemical localization of cytokeratin (red signal) in freshly prepared pseudobranch cells. FITC-labeled streptavidin was used to stain mitochondria (green signal). Three groups of cells were identified. The most intense staining was observed in small and largely undifferentiated cells (A,B). (A) Confocal images with three optical sections of the cells. The top image shows a x,z section, bottom left a x,y section, and bottom right a y,z section. Within each image the locations of the two accompanying sections are indicated by white cross-sectional bars, i.e. in the x,y section (bottom left) the horizontal bar indicates the location of the x,z section (top image), and the vertical bar the location of the y,z section (bottom right). (B) A three-dimensional reconstruction. (C) Cells from a small population of intermediate cell type, with the typical mitochondrial arrangement of pseudobranch cells, but smaller and more spheroid. These cells also stained positive for cytokeratins (cell marked with an arrow in C), but the staining was not as intense as in the undifferentiated cells. Apparently fully differentiated mitochondria-rich cells showed a very weak or no positive reaction. (D) A fully differentiated cell with a faint cytokeratin staining was observed. The staining only became visible at very high amplification of the red channel. In C and D, confocal images are shown with three optical sections of the cells. The top image shows a x,z section, bottom left a x,y section, and bottom right a y,z section. The locations of the sections within the cell cluster are indicated by white cross-sectional bars (see explanation to A). Scale bars, 10 µm.