Results In vivo immunolocalization of endocan In order to investigate the expression and localization of endocan in the aortic wall at days E12 and E14 (stages 38 and 40) of devel- opment when intimal thickening is distin- guishable and EndoMT occurs,18 serial paraffin sections were examined by confocal microscopy. A specific monoclonal antibody that recognizes epitopes on the N-terminus of endocan was used. At 12-14 days of development, the aortic wall is composed by the endothelium, which limits the vessel lumen, and radially oriented mes- enchymal cells originating from the endotheli- um and that constitute the intimal thickening. At these stages it is also possible to distinguish cells organized into circular lamellar and inter- lamellar layers that form the media. Occasionally, some mesenchymal cells and lamellar cell layers are observed displaying immunoreactivity for α-smooth muscle actin (α-SM actin) (Figure 1). At these stages, a strong immunoreactivity for endocan was found at the endothelium and some mesenchy- mal cells constituting the intimal thickening but not in the cells arranged in lamellar layers (Figure 1). No immunoreactivity was detected when the primary antibody was omitted or replaced by non-immune serum in control sections (not shown). In vitro immunolocalization of endocan Based on our findings in vivo, we also inves- tigated whether endocan was present in mono- layers of embryonic endothelial cells attached to FN that had been transiently mechanically altered during the explant removal, when they were deprived of serum for 2 h and switched to medium supplemented with EGF and ChS and maintained for an additional 10 or 12 h period. Immunofluorescence analyzed by confocal microscopy determined that in the EGF condi- tion in which separating, detaching, and migrating endothelial cells are observed and the vWf expression is notorious (Figure 2 a,b), all of the cells displayed strong immunoreactiv- ity for endocan (Figure 2 c,d). Specifically, this DSPG in addition to being located delineating the margins of some endothelial cells, was also located in punctate and linear arrays typical of focal complexes at the leading edge as well as in the area surrounding the microtubule- organizing center (MTOC) and aligned with microtubules (MTs) of separating, detaching, and migrating cells (compare panels c and e in Brief Note Figure 2). Focal adhesion complexes are con- sidered not only as structural and dynamic links between the ECM and the cytoskeleton controlling cell shape, spreading and motility, but also as sites for signal transduction through integrins and adaptor proteins such as focal adhesion kinase (FAK) and paxillin that are present in these sites. We therefore stud- ied the presence, organization and distribu- tion of these proteins by confocal microscopy in monolayers of embryonic endothelial cells attached to FN and stimulated with EGF for 10- 12 h. In such condition, immunofluorescence staining with anti-integrin-β3 and anti-p-FAK revealed that many cells displayed immunore- activity with a location and distribution resem- bling to that observed for endocan. Specifically, integrin-β3 and p-FAK appeared delineating the margins of some cells and organized into linear streaks and a punctate pattern at the leading edge of the cells that were separating, detaching, and migrating (Figure 3 a,b). In the SFM condition in which the endothelial cells displayed a cobblestone appearance and nei- ther separating, detaching nor migrating cells were observed, endocan, integrin-β3 and p-FAK Figure 2. Representative CLSM fluorescence images of vWf, endocan, and tubulin in monolayers of endothelial cells attached to FN after 10 h in culture in the presence of EGF. a) Strong immunoreactivity for vWf in a punctate and granular pattern typical of endothelial cells is observed in separating, detaching, and migrating cells. b) High mag- nification of vWf clustering around the nucleus of separating cells and disperse in the leading edge of migrating cells. c) Strong immunoreactivity for endocan is observed at the plasma membrane as well as in the leading edge and in the area surrounding the MTOC and aligned with MTs. d) Shows at high magnification the punctate and linear arrays of endocan. e) Tubulin immunoreactivity is observed surrounding the MTOC and in the MTs radially distributed along the cytoplasm. a), c), e) scale bars = 25 μm; b) and d) scale bars = 10 μm. [European Journal of Histochemistry 2011; 55:e13] [page 75]