93 Robert M. Hoffman (ed.), Multipotent Stem Cells of the Hair Follicle: Methods and Protocols, Methods in Molecular Biology, vol. 1453, DOI 10.1007/978-1-4939-3786-8_11, © Springer Science+Business Media New York 2016 Chapter 11 Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing Eduardo Martínez-Martínez, Eileen Uribe-Querol, Claudio I. Galván-Hernández, and Gabriel Gutiérrez-Ospina Abstract We describe a stereology method to obtain reliable estimates of the total number of proliferative and migra- tory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology. Key words Stereology , Hair follicles, Halogenated thymidine analogs, Iododeoxyuridine, Chlorodeoxyuridine, Bromodeoxyuridine, Nucleoside, Stem cells, Bulge, Epidermis, Wound, Keratinocytes, Random sampling, Confocal microscopy 1 Introduction One fundamental role of adult stem cells is to reestablish tissue integrity after injury by supplying new cells to the wounded area. In the skin epithelium, it is known that epidermal wounds activate the proliferation and differentiation of a group of stem cells that reside in an area of the hair follicle called the bulge [1–3]. The initial identification of the regions in the skin epithelium that con- tained putative stem cells was made possible by using tritiated thy- midine labeling and autoradiographic techniques. Using this method, the epithelial cells that retained the tritiated thymidine labeling, even for periods longer than 1 year, were found to be a population of cells (label-retaining cells) mainly located at the bulge area. These also display clonogenic properties in culture [1, 4]. Later identification of bulge-cell markers enabled their isolation and molecular characterization which led to the confirmation of the multipotency of these cells, both in vivo and in vitro [5–7].