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New Perspectives on Melanoma_ The Role of PAX3

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Unformatted text preview: ko) 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 microscope 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 procedures 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 40C 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. 75 Chapter III – Medic and Ziman, 2010 Acknowledgments The authors would like to thank Dr. Mark Brown for thoughtful discussions, Dr. Jennifer Thompson for technical assistance with microscopy and Dr. Robert White for constructive comments. The Pax3 monoclonal antibody, developed by C.P. Ordahl, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biolo...
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