Vector novared peroxidase solution

Paraffin-embedded tissue samples were dewaxed and treated for 5 min with proteinase K (Dako) for antigen retrieval. After blocking with 5 % skimmed milk in Tris-buffered saline (TBS), the primary antibody was applied. Before the incubation with the HRP-labelled secondary antibody, specimens were treated with peroxidase blocking solution (Agilent). For detection VECTOR NovaRED Peroxidase solution (Vector Laboratories) was used. Counterstaining was performed with 10 % Haemalaun-solution. The specimens were placed successively into 80 %, 96 %, and 100 % ethanol and finally in xylene before being mounted with Eukitt (Kindler). After photodocumentation, the cover slips and mounting medium were removed by placing the samples into xylene overnight. Sections were re-hydrated by descending xylene/alcohol series, de-stained in stripping buffer (2 % SDS, 0.8 % β-mercaptoethanol, 62.5 mM Tris-HCl pH=7.5) at 50°C for 1 h and washed consecutively in water, 95 % ethanol, water, and TBS. Antibodies are listed in Supplementary Table 2.

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.