Anti pd 1 antibody clone nat105

Protein expression patterns were assessed by immunohistochemistry using the following antibodies: anti-VEGFA (monoclonal, clone sp28, dilution, Spring Bioscience, California, USA), anti-CAIX (polyclonal, ab15086, dilution 1:1500, Abcam, Cambridge, UK), anti-PD1 (anti-PD-1 antibody, clone NAT105, dilution 1:50; Abcam, Cambridge, UK) and anti-PDL1 (anti-PD-L1 antibody, clone 130021, dilution 1:200; R&D Systems, Minneapolis, USA) [11] [12] [13] . The reactivity of antibodies was revealed with HRP-labelled polymer-conjugated secondary antibodies using diaminobenzidine (DAB) as the chromogen (Sigma-Aldrich, France). Negative controls were performed by omitting the primary antibody. The tumour expression for each antibody was independently evaluated (SFKJ and NRL) without A c c e p t e d m a n u s c r i p t knowledge of the case. The cut-off for positive cases was 30% of tumour cells for VEGF and 85% for CAIX as previously described [12, 14] . For PDL1, absent (0), weak (1), moderate (2) and strong expression (3) were reported and cases were then subdivided into negative (score 0-1) or positive (score 2-3) subgroups [15] . For PD1, immunostaining density was evaluated in tumour-infiltrating lymphocytes and was semi-quantified as absent, rare, moderate or dense as previously reported [15] .