79 Chapter 6 Limbal Epithelial Stem Cell Identification Using Immunoblotting Analysis Bernice Wright and Che J. Connon Abstract The unambiguous identification of limbal epithelial stem cells is currently a major challenge in corneal stem cell biology. Specific molecular markers which characterize these cells are lacking. At present, the best strategy for identification of limbal epithelial stem cells is to investigate a variety of putative markers for these cells in a differentiated (cytokeratin (CK) 3: CK3, integrin a6), undifferentiated (CK14), and naive state (ΔNp63 a, ATP-binding cassette subfamily G member 2 (ABCG2), integrin a9, Notch-1), alongside functional assays which indicate their stemness. The focus of this chapter is to highlight advances in the Western blotting technique for quantitative assessment of corneal epithelial cell markers, and the use of this technique for investigation of a range of different protein markers which identify limbal epithelial stem cells. Key words Limbal epithelial stem cells, Immunoblotting, Limbal epithelial stem cell markers, Western blotting, Quantitative Western blotting Adult stem cells reside in the outer limbal region of the cornea (Fig. 1), widely accepted as the palisades of Vogt [1, 2]. Emerging evidence suggests, however, that these cells are also present in lim- bal crypts [3]. Furthermore, their exclusive location in the limbal region of the cornea has been questioned [4]. Alongside the controversy of their specific location in the cor- nea, a prevailing problem in the study of limbal epithelial stem cells (LESC) is the lack of a definitive identifying marker [5]. The detec- tion of a range of cytokeratins in different regions of the cornea, by various methods including Western blotting, was previously reported to suggest the limbal location of epithelial stem cells [6]. A monoclonal antibody highly specific for a 64 kDa keratin, a marker for the advanced stages of corneal epithelial differentiation, demonstrated the presence of this marker in all layers of the cor- nea, but only in the suprabasal layers of the limbus. These reported 1 Introduction Bernice Wright and Che J. Connon (eds.), Corneal Regenerative Medicine: Methods and Protocols, Methods in Molecular Biology, vol. 1014, DOI 10.1007/978-1-62703-432-6_6, © Springer Science+Business Media New York 2013