334 Fig. 1 Expression of P2X5 and P2X7 receptors in human anagen hair follicles. a Longitudinal section of anagen hair follicle. P2X5 receptor immunostaining was present in the outer root sheath (ORS) and medulla (M) but not in the dermal papilla (DP). Scale bar=75 μm. b Transverse section of anagen hair follicle. P2X5 receptor immunostaining was present in both the inner root sheath (IRS) and the outer root sheath (ORS) but not in the cortex (CTX) or in the acellular medulla (M) of the hair shaft at this level. Scale bar=30 μm. c P2X7 receptor immunostaining (arrow) was present in the stratum corneum. There was no immunoreaction in the anagen hair follicle. Scale bar=75 μm. d P2X7 receptor immunostaining, transverse sec- tion. There was no staining in the outer root sheath (ORS), cortex (CTX) or acellular me- dulla (M) of the hair shaft at this level. Scale bar=30 μm. e, f Controls: the immunoreaction was abolished after preabsorp- tion of the e P2X5 and f P2X7 receptor antibodies with the corresponding peptides, con- firming the specificity of the immunoreaction. Scale bars=100 μm Purinergic Signalling (2008) 4:331–338 antibody (Fig. 2f) with the corresponding peptides, con- firming the specificity of the immunoreaction. Discussion In this study, we have obtained using immunohistochemis- try the first direct evidence for the expression of P2Y1, P2Y2 and P2X5 receptors in human anagen hair follicles. Double labelling of P2Y1 receptors with proliferation markers Ki-67 and PCNA showed that P2Y1 receptors were found in proliferating cells in the ORS and bulb in anagen hair follicles. The ORS is established during the early stages of anagen by the downwards migration of the regenerating epithelium and then maintains itself (in contrast to the IRS and hair shaft), independent of the bulbar matrix, by basal cell growth [21]. Thickness and cellularity of the ORS varies with the level of the follicle: it is single-layered near the bulb, higher up it is composed of multilayered cuboidal cells, and from the level of the sebaceous gland upwards it becomes multilayered and is structurally similar to the epidermis [22]. Previous work on normal interfollicular epidermis has shown that P2Y1 receptors show a strong immunopositive signal in the basal layer, and double labelling of P2Y1 receptors with keratinocyte proliferation markers Ki-67 and PCNA in the