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Unformatted text preview: staining overall with this polyclonal antibody (Invitrogen) compared to that observed with the monoclonal (DSHB). All of the samples analysed showed positive PAX3 staining in more than 10% of cells (Figure 3.1A, top panel). In normal skin PAX3‐positive cells showed a pattern of distribution that is characteristic of melanocytes; i.e. they were found at the epidermal‐dermal boundary and along the hair follicle (Figure 3.1A‐D). When adjacent sections were stained with MITF, a known marker of melanocytes, a comparable pattern of distribution was observed (Figure 3.1A, bottom panel). The intensity of PAX3 staining varied between cells within the same lesion and between different samples. There was no obvious correlation between either intensity or number of PAX3‐positive cells and lesion type. However, PAX3‐
56 Chapter III – Medic and Ziman, 2010 positive cells of normal skin samples generally showed weaker staining and far fewer cells were positive, compared to those in naevi and primary melanoma samples. In contrast, melanoma metastases have an overall low level of PAX3 expression and these cells were mainly located at the periphery of the lesion. Additional analysis by quantitative RT‐PCR was performed on a separate set of samples, to confirm PAX3 expression in normal skin, naevi, primary melanomas, and melanoma metastases. PAX3 expression was detected in all naevi (5/5), primary melanomas (5/5) and melanoma metastases analysed (5/5), and in one out of two (1/2) normal skin samples. The highest levels of PAX3 expression were found in melanoma metastases (mean fold change of 2583±1198), followed by naevi and primary melanomas with 101±39 and 73±55‐ fold change, respectively (Figure 3.1E). Even though these results suggest that PAX3 might be upregulated in advanced melanomas (melanoma metastases vs. primary melanomas) comparison with immunohistochemistry results show that expression levels are not due to increased amounts of PAX3 per individual cell, but rather they reflect the number of cells expressing PAX3 (Figure 3.1A). Nevertheless RT‐PCR results confirm PAX3 expressi...
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