332 trophic effects on these processes [3, 4]. Adenosine 5’- triphosphate (ATP) is now recognised as an important messenger molecule for cell–cell communication, with ATP binding specifically to purinergic receptors [5, 6]. Purinergic receptors are classified into two groups: P1 receptors are selective for adenosine, and P2 receptors are selective for ATP and adenosine 5’-diphosphate (ADP), which act as extracel- lular signalling molecules [6]. P2 receptors are divided into two main families: P2X receptors, which are ligand-gated ion channels, and P2Y receptors, which are G protein- coupled, based on molecular structure, transduction mecha- nisms and pharmacological properties [7]. Seven subtypes of P2X receptors [8] and eight subtypes of P2Y receptors are recognised [9]. There is growing evidence that ATP may act as an important local messenger in the epidermis. Purinergic receptors are expressed on rat cutaneous keratinocytes, and functional roles in the regulation of proliferation, differenti- ation and cell death have been proposed [10]. In particular, P2X5 receptors are expressed on cells undergoing pro- liferation and differentiation, whereas P2X7 receptors are associated with keratinised dead cells. P2Y2 receptors, found in the basal layer of normal epidermis, are claimed to be involved in keratinocyte proliferation [11]. P2Y1 receptors are thought to be mitogenic in endothelial cells [4]. Previous work on adult human interfollicular epidermis and primary keratinocyte cultures has suggested that P2Y1 and P2Y2 receptors are involved in keratinocyte proliferation and that P2X5 receptors are likely to be involved in keratinocyte differentiation, whereas P2X7 receptors are likely to be part of the machinery of end-stage terminal differentiation of keratinocytes [12]. This study demonstrates for the first time the distribution of P2X and P2Y receptors in human anagen hair follicles. We propose that these receptors are part of the normal homeostatic mechanisms controlling hair keratinocyte pro- liferation and differentiation. Materials and methods Tissues Eight samples of normal hair-bearing human skin were examined immunohistochemically in this study. Ethical Committee approval was obtained to harvest human skin. Samples of postoperatively redundant skin from otoplasty, preauricular skin tags and from the leg were obtained. Tissue was frozen in isopentane precooled in liquid nitrogen to cryoprotect the tissue and prevent damage to the tissue architecture from ice crystals. Blocks were sectioned at 10 µm on a cryostat (Reichert Jung CM1800), collected on gelatine-coated slides and air-dried at room temperature. The slides were stored at −20°C. Purinergic Signalling (2008) 4:331–338 Antibodies The immunogens used to produce polyclonal P2X5 and P2X7 antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracel- lular C termini of the cloned rat and human P2X receptors, as previously described [10, 13]. P2X5 and P2X7 antibodies (provided by Roche Bioscience, Palo Alto, CA, USA) were kept frozen at a stock concentration of 1 mg/ml. Polyclonal anti-P2Y1 and P2Y2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y1 (AA 242–258) and P2Y2 receptor (AA 227–244). Antibodies were kept frozen at a stock concentration of 0.6mg/ml (P2Y1, P2Y2). Proliferating cell nuclear antigen (PCNA) is a marker for proliferation in normal adult human keratinocytes [14]. Involucrin is a marker for keratinocyte differentiation [15]. PCNA (monoclonal antiproliferating cell nuclear antigen, clone PC10, raised in mouse ascites fluid; Sigma Chemical Co., Poole, UK) and involucrin (Sigma) antibodies were raised in the mouse. Ki-67 antigen is a marker for cell pro- liferation in normal human keratinocytes [16, 17]. Active caspase-3 is part of the apoptotic machinery of the cell and is expressed in terminally differentiating keratinocytes [18]. Ki-67 (DAKO, Heverlee, Denmark) and active caspase-3 (Abcam, Cambridge, UK) antibodies were both raised in the rabbit. Immunohistochemistry For immunostaining of cryostat sections, the avidin-biotin technique was used according to a revised protocol [19, 20]. Air-dried sections were fixed for 2 min in 4% form- aldehyde in 0.1M phosphate buffer containing 0.2% of a saturated solution of picric acid (pH 7.4). Fixation was carried out at this point, as earlier fixation of the entire tissue can result in alteration of the antibody binding sites, interfering with staining. Endogenous peroxidase was blocked for 10 min with 50% methanol containing 0.4% hydrogen peroxide. Nonspecific binding sites were blocked by a 20-min preincubation in 10% normal horse serum (NHS) in 0.1 M phosphate buffer containing 0.05% merthiolate (Sigma), followed by incubation with the primary antibodies diluted to 1:100 or 1:200 in antibody diluent [10% NHS in PBS + 2.5% sodium chloride (NaCl)] at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit immunoglobulin G (IgG) (Jackson Immu- noResearch Lab, West Grove, PA, USA) diluted to 1:500 in 1% NHS in PBS for 30 min, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1000 in PBS for 30 min at room temperature. After a wash step, a nickel- diaminobenzidine (DAB) enhancement technique was used to visualise the reaction product. Sections were washed three