To avoid bias, two of the authors (JZ and VM) scored all specimens independently and blindly, and the results were averaged. For spatial quantification, limbal demarcation lines were drawn based on the nuclear appearance of basal epithelial cells indicated by DAPI stain (see Results). circumference, within an annular zone of 100200 m wide. LRC density was highest in Trimebutine maleate the superior temporal quadrant and lowest in the inferior nasal quadrant. These results show that LRCs are present asymmetrically in a narrow zone within the limbus that can be defined precisely in reference to a newly defined anatomical boundary line between the cornea and the limbus.(J Histochem Cytochem 57:177185, 2009) Keywords:limbus, corneal epithelium, epithelial homeostasis, label-retaining cells, nuclear staining Thecornealepitheliumis a self-renewing tissue, and its stem cells are thought to reside primarily in the basal layer of the limbus, a transitional zone between the cornea and the conjunctiva (Dua and Azuara-Blanco 2000;Lavker et al. 2004;Wolosin et al. 2004;Pajoohesh-Ganji and Stepp 2005;Schlotzer-Schrehardt and Kruse 2005). The prevailing idea is that differentiated daughter cells are generated through asymmetrical divisions of stem cells in the limbus, enter the cornea, and move centripetally (Buck 1985;Nagasaki and Zhao 2003) and vertically (Beebe and Masters 1996) as part of epithelial homeostasis (Thoft and Friend 1983). As such, the importance of stem cells as the ultimate source of the tissue has been recognized, but there have been no reports of identifying individual limbal stem cells in situ. In the absence of definitive biochemical markers (Pajoohesh-Ganji and Stepp 2005;Schlotzer-Schrehardt and Kruse 2005), epithelial stem cells are frequently described as part of a population of slow cycling, label-retaining cells (LRCs), on the premise that stem cells divide less frequently than other differentiated cells (Bickenbach 1981;Cotsarelis et al. 1989). Although it Trimebutine maleate is clear that all LRCs are not stem cells (Braun and Watt 2004), the nearly exclusive presence of LRCs in the limbus has been regarded as one of the evidence that stem cells are enriched in the limbus (Cotsarelis et al. 1989;Lehrer et al. 1998;Lavker et al. 2004). Previous studies suggest that stem cells are not evenly distributed along the limbus in human (Wiley et al. 1991;Pellegrini et al. 1999;Shanmuganathan et al. 2007) and mouse eyes (Pajoohesh-Ganji et al. 2006), but specific location of stem cells within the limbus against a known marker has not been reported. One of the difficulties of determining stem cell location within the limbus is that boundaries of the limbus itself are not well defined (Van Buskirk 1989). In the mouse eye, an apparent lack of Bowman’s membrane makes it even harder to draw the anatomical limbus. It seems necessary to first identify Trimebutine maleate a universal structural marker in or in the immediate vicinity of the limbus that is present in all eyes, so that stem cell location could be referenced against it. Two of the major structural components in the limbus are capillary vessel arcades and stromal nerve trunks, but neither seems to be suited as a fiduciary marker because of heterogeneity of their patterns in different eyes. Given this background, we set out to determine the precise location of LRCs in the limbus of a mouse eye, in the hope that this information will assist studies on limbal stem cells, including a search for their molecular markers. Our results show that an anatomical boundary between the cornea and the limbus could be determined reliably by a histological method and that this boundary line could be used as a fiduciary marker to describe the spatial distribution of individual LRCs. == Materials and Methods == == Animals == All animal procedures were approved FLJ25987 by the Columbia University Institutional Animal Care and Use Committee. CAG-EGFP mice [ubiquitous green fluorescent protein (GFP), Trimebutine maleate stock number 003115] were obtained from the Jackson Laboratory (Bar Harbor, ME), and a colony was maintained in-house. Only male mice were used in this study. We chose to study this line because we have accumulated a body of quantitative data on epithelial cell movement and cell division in the ocular surface of these mice (Nagasaki and Zhao 2003,2005; unpublished data), and we intend to further analyze epithelial homeostasis with them. Histone H2B-EGFP mice (stock number 005418; Jackson Laboratory) were used in.