Automated pt link

Prior to antibody staining and antigen retrieval, slides were baked at 60°C for 60 min. Deparaffinisation and antigen retrieval was performed using an automated PT link (Dako, heat-mediated antigen retrieval at high pH). An Autostainer Link 48 (Dako) was used to perform antibody staining using an EnVision FLEX visualisation system. Details of antibodies and their working dilutions are shown in supplementary table S1. Using protocols provided by the manufacturer, antibody binding was visualised using a FLEX 3,3′-diaminobenzidine (DAB) substrate working solution and haematoxylin counterstain (Dako). After staining, slides were taken through graded industrial methylated spirit and xylene and mounted in Pertex mounting medium (Histolab). Isotype control staining was performed to confirm specificity of staining, whereby the primary antibody was substituted for universal isotype control antibodies. The murine universal isotype control used was a cocktail of mouse IgG1, IgG2a, IgG2b, IgG3 and IgM (Dako IR750). The universal isotype control for rabbits was an immunoglobulin fraction of serum from non-immunised rabbits, solid-phase absorbed (Dako IR600).

For processing of tissue, see Additional file 1. Sections were pre-treated and deparaffinized in an automated PT link (Dako, Glostrup, Denmark) according to Additional file 1: Table S2.