Mouse anti hscd163

FFPE melanoma sections were prepared as described before. Blocking was performed with 5% donkey serum in PBS. Primary antibodies [self-generated rabbit anti-hsP2Y12 and mouse anti-hsCD163 (Leica, Wetzlar, Germany)] were incubated at 4 °C overnight. Subsequently, the samples were treated with the corresponding fluorochrome-conjugated secondary antibody [donkey anti-rabbit Alexa488 and donkey anti-mouse Cy3 (both Dianova, Hamburg, Germany)]. Images were taken with the Nikon Eclipse NI microscope with the Clara interline CCD camera (Andor, Belfast, UK) and NIS-Elements Advanced software (Nikon, Düsseldorf, Germany).

FFPE melanoma sections were prepared as described before. Samples were blocked with 5% skimmed milk powder in PBS and incubated with the desired primary antibody [self-generated rabbit anti-hsP2Y12, mouse anti-hsCD163 (Leica, Wetzlar, Germany), mouse anti-hsCD68 (Dako by Agilent, Waldbronn, Germany)] diluted in antibody diluent (Dako by Agilent, Waldbronn, Germany) either for 2 h at RT or overnight at 4 °C. The samples were incubated with peroxidase blocking solution (Dako by Agilent, Waldbronn, Germany) followed by incubation with the appropriate HRP-labeled secondary antibody diluted in antibody diluent (Dako by Agilent, Waldbronn, Germany) for 1 h at RT. Samples were incubated with VECTOR NovaRED Peroxidase Solution (Vector laboratories, Peterborough, UK) and counterstained using 10% Mayer’s Haemalaun (Merck, Darmstadt, Germany). After pictures were taken with the Nikon Eclipse NI microscope specimens were destained in stripping buffer [2% SDS, 62.5 mM Tris-HCL (pH 7.5), 0.8% ß-mercaptoethanol] for 1 h at 50 °C and used again to stain another antigen.