Purinergic Signalling (2008) 4:331–338 times with PBS after each of the above steps except for after preincubation with 10% NHS. After the last wash, sections were dehydrated twice in isopropanol and mounted with EUKITT (BDH Laboratory Supplies, Poole, UK). Control experiments were carried out with primary antibodies omit- ted from the staining procedure or the primary antibodies preabsorbed with the corresponding peptides. Double labelling of P2X or P2Y receptor antibodies with either PCNA or involucrin Results 333 Photography The results were photographed using a Zeiss Axioplan high-definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland). Sections were fixed and incubated with P2X or P2Y anti- bodies overnight, as described above. After a wash step, biotinylated donkey anti-rabbit IgG antibody, diluted 1:500 in 1% NHS in PBS, was then applied for 1 h, followed by Streptavidin Texas red (Amersham Plc., Little Chalfont, UK), diluted 1:200 in PBS-merthiolate for 1 h at room temperature. Sections were preincubated for 30 min with 10% normal goat serum (NGS) diluted in 0.1 M phosphate buffer containing 0.05% merthiolate (Sigma). They were then incubated for 2 h at room temperature with one of the following antibodies: PCNA antibody (Sigma) diluted 1:1000; mouse monoclonal anti-involucrin (Sigma) diluted 1:50. After a wash step, the directly labelled secondary antibody goat anti-mouse FITC (Nordic Immunological Laboratories, Tilburg, The Nether- lands) was applied at a dilution of 1:200 for 1 h, and sections were washed and mounted in Citifluor (Citifluor Ltd., Leicester, UK). P2X or P2Y receptor immunostaining ap- peared red and PCNA and involucrin stained green. Double-labelling of P2X or P2Y receptor antibodies with either Ki-67 antigen or anti-human caspase-3 Sections were fixed and incubated with P2X or P2Y antibodies overnight, as described above. After washing, sections were incubated with biotinylated donkey anti- rabbit IgG (Jackson) diluted to 1:500 in 1% NHS in PBS for 1 h, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1500 in PBS for 1 h, tyramide amplification for 8 min (Tyramide Amplification Kit, NEN Life Science Products, Boston, MA, USA) and then Streptavidin Texas red (Amersham), diluted 1:200 in PBS-merthiolate for 10 min. Sections were washed three times in PBS after each of the above steps. Sections were preincubated for 20 min in 10% NGS and incubated at room temperature for 2 h with one of the following antibodies: rabbit anti-human Ki-67 antigen (DAKO) 1:50 or rabbit anti-human active caspase-3 (Abcam). Sections were washed and incubated with the directly labelled secondary antibody Oregon-green-labelled goat anti-rabbit IgG (Jackson) diluted 1:100 for 45 min. Sections were then washed and mounted in Citifluor (Citifluor). P2X or P2Y receptor immunostaining appeared red; Ki-67 and caspase-3 stained green. Only hair follicles in the anagen (growth) phase were studied, as hair follicles in catagen (regression) or telogen (resting) phases of the hair cycle were not seen in the sections analysed. P2X5, P2X7, P2Y1 and P2Y2 receptors are expressed in human anagen hair follicles P2X5 receptor immunoreactivity was observed in the hair keratinocytes of all human skin samples. P2X5 receptor staining was restricted to the medulla and both the IRS and ORS (Fig. 1a,b), which were continuous with the surface epidermis. P2X7 receptor immunostaining was present in the stratum corneum of the overlying epidermis, but there was no staining in the anagen hair follicle (Fig. 1c,d). P2Y1 receptors were found in the ORS and bulb of anagen hair follicles but not in the IRS (Fig. 2a,b). P2Y2 receptors were found in cells in the cortex, with possibly a few positive cells at the edge of the medulla, but no staining in the central medulla (Fig. 2c,d). Double labelling of P2Y1 receptors with Ki-67 showed that P2Y1 receptors were found in proliferating cells in the ORS and bulb of anagen hair follicles (Fig. 3a,b). Double labelling of P2Y2 receptors with PCNA showed that P2Y2 receptors were expressed in the cortex and possibly at the edge of the medulla (Fig. 3c) and PCNA was expressed in the ORS and bulb (Fig. 3c,d). P2Y2 receptors were only expressed in viable cells and not in the keratinised hair shaft or the central medulla (Fig. 3d). Involucrin was expressed both in the cortex and IRS but not in the ORS (Fig. 3e,f). P2X5 receptors were expressed in the medulla, IRS and ORS. There was colocalisation of involucrin with P2X5 receptor staining in the IRS and in a few cell layers at the edge of the medulla (Fig. 3e,f). Double labelling of P2X7 receptors with active caspase-3 did not show any immunoreaction within the anagen hair follicle. Control immunostaining experiments Both omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. The immunoreaction was abolished after preabsorption of the P2X5 (Fig. 1e), P2X7 (Fig. 1f), P2Y1 (Fig. 2e) or P2Y2