Sc 13117

Equal amounts of protein were subjected to electrophoresis using Mini-Protean TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA) and then transferred onto polyvinyl fluoride membrane. Blocking was performed with 5% non-fat milk in Tris-Buffered Saline. Primary antibody incubations were carried out at +4°C for overnight (NRP1 at dilution 1:1,500, ab81321, Abcam; NRP2 at dilution 1:800, sc-13117, Santa Cruz). Secondary antibody incubation was performed at room temperature for 1 h (1:10,000; #115-005-062 or #111-035-144, Jackson ImmunoResearch, West Grove, PA, USA). Protein bands were illuminated utilizing the Enhanced Chemiluminescence detection kit (Amersham ECL reagent; GE Healthcare, Barrington, IL). Quantification was performed with Image Lab Software 6.0 (Bio-Rad). NRP1 and NRP2 band intensities were normalized to amount of total protein in corresponding lane (Supplementary Figure 1) utilizing stain-free technology (37 (link)).

FFPE samples were cut to 5-μm sections and deparaffinized with xylene. For antigen epitope unmasking, samples were treated with antigen target retrieval solution (pH 9; Dako, Glostrup, Denmark) for 30 min at +98°C. Next, endogenous peroxidase activity was blocked with 3% hydrogen peroxidase and non-specific binding was averted with 0.4% casein (both solutions from Novolink Polymer Detection System Kit; Leica, Newcastle, UK). Primary antibody incubations were performed either at +4°C for overnight (NRP2 at dilution 1:2,000; sc-13117, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or at room temperature for 1 h (NRP1 at dilution 1:3,000; ab81321, Abcam, Cambridge, MA). Antibody binding was visualized with polymerized reporter enzyme staining system (Novolink Polymer Detection System Kit). Positive LSEC staining was used as an internal control for both antibodies. Immunoreactivity was scored based on intensity (negative, low/intermediate, or high) by two separate observers. Imaging was performed using 3DHISTECH Panoramic 250 FLASH II digital slide scanner at Genome Biology Unit (Research Programs Unit, Faculty of Medicine, University of Helsinki Biocenter, Helsinki, Finland).