C6219

Human melanoma samples and normal controls were provided by the Ontario Tumor Bank, which is funded by the Ontario Institute of Cancer Research (OICR). All specimens were verified by a pathologist at the OICR before use. Paraffin embedded sections (5 µm) of primary cutaneous tumor (N = 14), nodal melanoma metastases (N = 15), and melanoma metastases from distant organ sites (N = 7), in addition to normal controls (N = 7) were selected. All work with human specimens was approved by The Human Science Research Ethics Board at the University of Western Ontario. Paraffin embedded sections were deparaffinized, subjected to antigen retrieval, and probed with rabbit anti-Cx43 (1:300; Sigma-Aldrich, C6219) or rabbit anti-MITF (1:200; Abcam, 20663) antibodies, overnight at 4°C prior to labeling with secondary antibodies as described above. To avoid the possible inclusion of adjacent normal tissue in analysis, 5-10 images of the tumor core and its surrounding radius of all samples was imaged using the 40x objective of the Zeiss LSM 800 confocal microscope. Images were then classified relatively as possessing no, low or high intensity of Cx43, and plaques were identified as punctate structures (0.2-1 μm in size) at the interface of two adjacent cells. Tissues were imaged blinded to the investigator to mitigate bias.