Pt link module

Multi-tissue arrays (TMA) containing two 1.5-mm representative punches of 62 TH and TC patient samples were used for immunohistochemical (IHC) stainings (Table 1). All antibodies were established on positive control tissues chosen from the Human Protein Atlas (http://www.proteinatlas.org). Stainings were performed on 2-μm sections according to a standard protocol on an Autostainer (Agilent, USA). In brief, antigen retrieval was performed at 95°C in pH 6 or pH 9 Envision FLEX target retrieval solution in a PT Link Module (Agilent, USA) followed by 1-h incubation with primary antibodies (Additional file: Table S2). Samples were washed with PBS and incubated with an appropriate secondary antibody (EnVision Flex+, Dako) for 30min. Two individual observers (DM and PS) evaluated stainings for both cores of a respective case and graded as positive when >50% of the tumor cells were positive. Staining intensity was scored 0 to 2 (0, negative staining; 1, weak staining; 2, strong staining). The average staining intensity of two cores was taken for further analysis. To evaluate the best clinical separation and to define the optimal threshold for dividing IHC low and high staining intensities, the cutoff finder was used as described by Budczies et al. [33 (link)]. These resulting cutoff values are given in the corresponding figure legends.

We evaluated previously reported IHC findings of melanocytic markers, including S-100 protein, Melan-A, HMB45, and SOX-10, and assessed their positivity. We also performed additional IHC using representative sections from formalin-fixed and paraffin-embedded tissues in some cases. These tissues were sliced into 3-mm-thick sections and examined with an automated IHC system at Sapporo Medical University Hospital. All slides were loaded into a PT Link module (Agilent Technologies, Santa Clara, CA) and subjected to a heat-induced antigen-retrieval protocol with EnVision FLEX Target Retrieval Solution (Agilent Technologies) before being transferred to the Autostainer Link 48 instrument (Agilent Technologies) and Dako Omnis (Agilent Technologies). We used antibodies against the following antigens: S-100 protein (polyclonal; Agilent Technologies), Melan-A (A103; Agilent Technologies), HMB45 (HMB45; Agilent Technologies), and SOX-10 (N-20; Santa Cruz Biotechnology, Santa Cruz, CA).