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Unformatted text preview: analysed their co‐expression with PAX3 in normal skin melanocytes, naevi and melanomas. Interestingly, while the majority of PAX3‐positive melanocytes in the epidermis of normal skin show a mature phenotype, a portion (around 20%) show a less differentiated and proliferative phenotype. Moreover, PAX3‐positive melanocytes frequently show expression of BCL2‐like 1 (BCL2L1, also known as Bcl‐X, bcl‐xL or BCL‐XL/S), an antiapoptotic marker. Taken together these results indicate that melanocytes of the normal skin display a phenotype that is predisposed to malignant transformation (antiapoptotic, undifferentiated and proliferative). We also show that PAX3‐positive epidermal melanocytes of normal skin are molecularly distinct from those of the hair follicle, which like melanoma cells 55 Chapter III – Medic and Ziman, 2010 express Melanoma cell adhesion molecule (MCAM, also known as MUC18/CD146), associated with cell migration and melanoma progression and metastasis. Results PAX3 expression in normal skin, naevi and melanoma PAX3 expression was analysed by immunohistochemistry in tissue sections from paraffin embedded samples of normal skin, naevus, primary melanoma and melanoma metastases. Positive staining for PAX3 was obtained after performing heat‐induced antigen retrieval in an EDTA/Tris buffer, pH 8.0 and incubating the primary antibody (anti‐PAX3, DSHB) at room temperature (Figure 3.1A, top panel). Antigen retrieval using the most common 10mM Citrate buffer, pH 6.0 with the same staining protocol, failed to produce a positive signal in several of these samples, and in those that were positive after citrate antigen retrieval, the intensity of staining was dramatically reduced. Negative controls included omitting the primary antibody from the procedure resulting in no immunohistochemical signal (not shown). Mouse monoclonal anti‐PAX3 antibody (DSHB) has been previously confirmed by immunohistochemistry to be specific to PAX3 protein . PAX3‐specific staining was also confirmed using a different commercially available anti‐PAX3 antibody (Invitrogen) (Figure 3.2, A‐C and E). However, we have observed less intensive...
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- Spring '14