code 07-189 MEDIC). Written consent was obtained for the use of patient archival tissue samples. Samples were previously diagnosed by certified pathologists. 4mm thick formalin fixed paraffin embedded tissue sections were used for immunohistochemistry and immunofluorescence. The following samples were analysed by immunohistochemistry and immunofluorescence: 10 normal skins (including 3 hairy, 2 limb and 2 breast skin samples, and 3 samples had no anatomic location specified), 14 naevi (including 5 intradermal, 6 compound and 3 junctional samples), 15 primary (including 8 superficial spreading melanoma samples, 2 of radial growth phase, and 1 sample each of acral lentiginous, nodular, neurotropic, recurrent and lentigo maligna melanoma) and 5 systemic melanoma metastases (2 small bowel and 3 lymph node). Tissue samples characterized here as normal skins were free of any melanocytic lesion, having been used for other diagnostic purposes (solar keratosis, seborrhoeic keratosis and granulomatous follicu- litis) or tissue obtained after breast reduction. Normal skin samples originated mainly from limbs or face (referred to as sun-exposed skin) or breast (referred to as reduced sun-exposed skin). Additionally, a separate set of 12 archival samples, including 2 normal skin, 5 naevi and 5 primary melanomas as well as 5 cryopreserved lymph node melanoma metastases were analysed by qRT-PCR. RNA extraction and qRT-PCT Total RNA was extracted from the paraffin embedded and cryopreserved tissue samples using Aurum Total RNA Mini Kit (Bio-Rad) according to the manufacturer’s recommendations. Slight modifications were made to the protocol used for paraffin embedded samples. Mainly, sections were dewaxed in Xylene and absolute Ethanol (each twice for 10 minutes), before tissue was scratched from the slides and lysed in a mixture of Tissue Lysis Buffer (High Pure RNA Paraffin Kit, Roche), Proteinase K and 10% SDS by incubation at 55uC overnight. The quality and integrity of extracted RNA was assessed by gel electrophoresis and subsequent amplification of the housekeeping gene GAPDH. 250ng of the total RNA was reverse transcribed with Omniscript RT kit (Qiagen), according to the manufacturers’ instructions and PCR was performed using Taq DNA Polymerase (Qiagen). Primers used were, for GAPDH: 59-GGG TGT GAA CCA TGA GAA GT-39 (forward) and 59-GAC TGT GGT CAT GAG TCC T-39 (reverse) (Takeuchi et al. 2003); and for PAX3: 59- AGT GAG ATT ACG CGC TAG -39 (forward) and 59-CCA GCG GAA GAC CAG AAA C-39 (reverse). Real time PCR was performed using iQ SYBR Green Supermix (Bio-Rad). Each sample was run in duplicate and assays included negative controls (reagents without RNA or cDNA) and positive controls (plasmid DNA containing a PAX3 insert). The calibration curve was generated using the threshold cycle (Ct) of 8 serial dilutions of plasmid DNA template with known copy number. The Ct value of the sample was interpolated from the standard curve and mRNA copy number, mean value and standard deviation were calculated using iQ5 RealTime Detection System Software (Bio-Rad Laboratories). The increase in the mean values of mRNA copy number for each sample relative to that in the normal skin was expressed as a fold-change increase in mRNA. Antibodies The following primary antibodies were used: mouse monoclonal to PAX3 (DSHB, 1/10), rabbit polyclonal to PAX3 (Invitrogen, 1/ 500), mouse monoclonal to MITF (Merck, 1/20), mouse monoclonal to MLANA (Merck, 1/50), rabbit polyclonal to HES1 (Abcam, 1/100), rabbit monoclonal to BCL2L1 (Abcam, 1/ 50), rabbit monoclonal to MCAM (Abcam, 1/500) and rabbit PAX3 in Melanocytes polyclonal to Ki67 (Abcam, 1/25). For immunofluorescent staining the following secondary and tertiary antibodies were used: anti-mouse conjugated AlexaFluor-488 (1/500), anti-rabbit conjugated AlexaFluor-488 (1/500), anti-mouse conjugated Alex- aFluor-546 (1/500), or biotinylated anti-rabbit IgG (1/500) linked to streptavidin-conjugated AlexaFluor-546 (1/500). All antibodies were diluted in PBST (0.2% TritonX-100 in PBS) together with 1%NGS for immunofluorescent staining. Immunohistochemistry Paraffin embedded sections were dewaxed in xylene (365 min- utes) and dehydrated in ethanol series (365 minutes in 100% ethanol, 1 minute in each of 95% and 70% ethanol, and 3610 dips in ddH20). Antigen retrieval was routinely performed in EDTA/Tris, pH 8.0, by heating for 365 minutes in a microwave oven, at approximately 750 Watts. Slides were left to cool to room temperature for at least 20 minutes. Sections were washed in PBS and endogenous peroxidases were blocked with 3% H202 for 10 minutes. After rinsing in PBS, sections were blocked with 10% FCS for one hour, followed by incubation with primary antibodies for one hour at room temperature. After washing in PBS, the secondary antibody linked to biotin (Dako, LSAB kit) was applied and sections were incubated for 20 minutes at room temperature. After washing in PBS-Tween20 (0.05%), sections were incubated with streptavidin conjugated horseradish perox- idase (Dako, LSAB kit) for 20 minutes at room temperature, washed in PBS-Tween20, then incubated with DAB (Dako) for several minutes. The reaction was stopped by washing in ddH20, and the signal was enhanced by applying a solution of CuSO4 and NaCl, for 3 minutes. Hematoxylin staining was performed before the slides were mounted with DPX neutral mounting medium. Results were analysed on an Olympus BX51 micro- scope and images were captured using an Olympus DP71 camera. Controls with primary antibodies withheld were immunonegative. The specific staining of nuclear markers PAX3 and MITF was generally distinguishable from the cytoplasmic melanin deposit in the tissue by their sub-cellular localisation. Intensity of PAX3 staining was compared across all samples analysed and described as either weak or strong. Immunofluorescence The same dewaxing, rehydration and antigen retrieval proce- dures described above were followed for immunofluorescence. Sections were blocked with 10%NGS for one hour at room temperature, followed by incubation with primary antibodies at 4uC overnight (for rabbit PAX3 and MITF co-staining) or at room temperature for one hour (for all other antibodies). After washing in PBS, sections were incubated with the appropriate secondary antibody (followed by tertiary only for rabbit PAX3 and HES1) for one hour and washed in PBS. For counterstaining Hoechst 33342 was used. Sections were mounted with FluorSave Reagent (Calbiochem) and analysed on an epifluorescent Olympus BX51 microscope equipped with an Olympus DP71 camera. Controls with primary antibodies withheld were immunonegative. For each sample, quantification of cells positive for a given marker was performed by analysis of 5–10 representative regions of the section. The number of double-labelled cells was calculated relative to the number of cells positive for a comparative marker and expressed as a percentage. Acknowledgments The authors would like to thank Dr. Mark Brown for thoughtful discussions, Dr. Jennifer Thompson for technical assistance with PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e9977