Qualitative detection of tumor-specific antibodies by immunocytochemistry was performed by centrifugation of 2610 5 cells at 300g using a Shandon CytospinH 4 Cytocentrifuge (Thermo Fisher Scientific, Waltham, MA) onto glass slides. Cells were fixed in 0.5% formalin and antibodies, such as those recognizing the human High Molecular Weight Melanoma-Associated Antigen (anti-HMW-MAA clone LHM2, Invitrogen, Carlsbad, CA), were incubated overnight at 4uC and detected following a 2 hour incubation at 4uC with a horseradish peroxidase-conjugated anti-IgG Fc-specific antibody (1:100 dilution in Tris Buffered Saline, Sigma, Dorset, UK). Slides were stained with DAB chromogenic substrate (DAKO, Ely, UK) for 5 minutes, washed and counterstained with Mayer's hematoxlin (Merck, Darmstadt, Germany) for one minute, dehydrated and mounted in DPX mountant (Sigma) prior to assessments.