Dako link platform

FFPE 3-µm sections were obtained from paraffin-embedded human tumor xenografts in mice and human tumors, and IHC was performed as previously described [50 (link)]. Briefly, FFPE sections were placed on positively charged glass slides on a Dako Link platform (Dako, Agilent Technologies Spain, Madrid, Spain). After deparaffinization, heat antigen retrieval was performed in a pH 9 EDTA-based buffered solution (Dako). Endogenous peroxidase was quenched. Primary antibodies (all from Cell Signaling) against YAP1 (#14074), TAZ (#1674883/v386), cleaved caspase3 (c-casp3) (1:100), and phospho-Histone3-Ser10 (pH3) (1:100) were used, and HER2 status was assessed by HercepTest (K5207; Dako). Antigen–antibody reaction was detected by incubation with an anti-mouse/rabbit Ig-dextran polymer coupled with peroxidase (Flex+, Dako). Sections were then visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin. All immunohistochemical staining was performed on a Dako Autostainer platform. A semiquantitative estimation of the percentage of tumor cells positively stained for YAP1 and TAZ nuclear expression was calculated by a pathologist (FR), and the results ranged from 0 to 100. Proper positive and negative controls were assayed to validate the staining procedure (Figure S16).