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Unformatted text preview: observed primarily when using rabbit polyclonal antibody to PAX3 (Invitrogen) for immunofluorescent staining. When the mouse monoclonal anti‐PAX3 antibody (DSHB) was used for immunohistochemistry staining, many more PAX3‐positive cells were observed on the same sample. The discrepancy between these two results is probably due to the sensitivity of the two antibodies. Nevertheless, the intensity of PAX3 staining varied when either antibody was used, whereas MITF levels were uniform across samples (Figure 3.2, A‐C). This is shown clearly in the A2058 metastatic cell line (Figure 3.2C) and may also reflect different cell status/phenotype. 59 Chapter III – Medic and Ziman, 2010 Figure 3.2. Coexpression of PAX3 and MITF in melanocytic and melanoma cells. A‐
C) Double immunofluorescent staining showing co‐expression of PAX3 and MITF in: (A) epidermal melanocytes of normal skin; (B) primary melanoma; and (C) in an A2058 metastatic melanoma cell line. Arrowheads in (A) show PAX3‐positive normal epidermal melanocytes. Lines in (A) and (B) demarcate epidermal‐dermal border (EDB). The variable PAX3 expression was clearly visible in A2058 cell line (C). For all these experiments depicted in the figure, PAX3 was labelled with rabbit polyclonal antibody (Invitrogen). D) Graph showing overall number of MITF and PAX3 double labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of MITF‐positive cells that are also PAX3‐positive averaged across all samples. E) Double immunofluorescent staining shows PAX3 and MITF co‐expressing melanocytes (yellow‐orange) in the bulb of the hair follicle of normal skin. Note the single MITF‐
labelled melanocytes (green) at the base of the hair. 60 Chapter III – Medic and Ziman, 2010 PAX3 in melanocyte differentiation: Coexpression of PAX3 with MITF, HES1 and MLANA Even though the overall percentage of MITF‐positive melanocytes that were also PAX3‐positive in normal skin was not significantly different to either...
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This document was uploaded on 03/06/2014 for the course CHEMISTRY 12 at National University of Singapore.
- Spring '14