Immunofluorescence and Immunohistochemistry Protocol

For IC analysis, SKMel103, SKMel147, SKMel28 and UACC903 cells were seeded directly on cover slides in a 24-well plate (Sarstedt, Nümbrecht, Germany) at 50% confluence, incubated overnight and subjected to treatment the next day. Cell staining was performed as previously described^{54 (link)}. For IF and IHC, 4 µm sections of formalin-fixed paraffin-embedded tumor samples were subjected to immunocytochemistry according to the manufacturer’s antibody protocol. The samples were developed by using either secondary antibodies linked to horseradish peroxidase with the UltraView^TM Universal DAB Detection Kit (Ventana Medical Systems) or secondary antibodies linked to fluorophores. Staining was performed either manually or on the automated immunostainer Beckmarck XT (Ventana Medical Systems, Roche, Tucson, AZ, USA). For the samples processed manually, antigen retrieval was performed using target retrieval solution pH 6.0 (Agilent). The samples were scanned (panoramic slide digital scanner) and evaluated by two independent pathologists (using 3DHistech software).