Envision flex wash buffer

Immunostainings were carried out on 3 μm-thick tissue sections according to standard procedures. Briefly, after antigen retrieval, samples were blocked with Peroxidase-Blocking Solution (Dako, S202386) for 10 min at RT. Primary antibodies (please refer to Supplementary Table 3) were incubated o/n at 4 °C. Slides were washed with EnVision™ FLEX Wash Buffer (Dako, K800721). Secondary antibody was incubated for 45 min at RT. For anti-rat Biotin Donkey IgG, additional 20 min incubation at RT with streptavidin HRP (Sigma, S2438, 1/1000 dilution) was performed. Samples were developed using 3,3′-diaminobenzidine, counterstained with hematoxylin and mounted. Staining analyses were performed with either QuPath software v.0.3.2^{76 (link)} or H-Score^{77 (link)}. In more details, H-Score representing overall stromal POSTN protein levels was assessed for each sample by expert pathologist using intensity scores (from 0 to 3) and intensity scores frequencies (from 0 to 100%). H-Score was calculated as follow: (% of scored 1 stromal area) × 1 + (% of scored 2 stromal area) × 2 + (% of scored 3 stromal area) × 3.

Tissues were deparaffinized and rehydrated to water, and after a low-temperature retrieval technique, ALTER,^{24 (link)} they were immunostained on a Dako Autostainer (Dako, Stockport, UK). Briefly, staining comprised a 10-minute endogenous peroxidase block (Dako) followed by a 10-minute protein block in 2% casein (Vector Labs, Birmingham, UK). Sections were incubated in optimally diluted antibodies for 1 hour; mouse anti-TNFα, 1/200 (sc52746, Santa Cruz Biotechnology, CA, USA) and rabbit anti-integrin b7, 1/400 (HPA042277; Sigma-Aldrich, Gillingham, UK). Antibody detection was performed with Vector Excel mouse and rabbit kits, respectively and visualized in NovaRED chromagen (Vector Labs) for 5 minutes. All buffer washes performed were with EnVision FLEX wash buffer (Dako). Sections were then counterstained with Meyers hematoxylin, dehydrated through to xylene, and mounted with a glass coverslip in distyrene plasticizer xylene. Expression was scored as the following: 0 = none, 1 = low (<30%), 2 = medium (30%-60%), and 3 = high (>60%).

Antigen expression of Survivin and Mucin-1 was assessed on archived FFPE blocks of tumor biopsies taken from metastatic lesions. Mucin-1 staining was performed according to a protocol described previously (17 (link)). For Survivin, the procedure was similar. First, 4-μm thickness sections were cut, and slides were deparaffinized and rehydrated prior to antigen retrieval by boiling in EnVision™ FLEX target retrieval solution (pH 9, K8004, Dako) for 10 minutes. Subsequently, endogenous peroxidase was blocked using 3% hydrogen peroxidase (76,051,800.1000, EMD Millipore) in PBS (4391.9010, Klinipath) for 10 minutes. Incubation was performed with a primary anti-Survivin antibody (D8, sc-17779, Santa Cruz Biotechnology, TX, USA, dilution) for 1 hour at room temperature. EnVision™ FLEX Wash Buffer (DM831, Dako) was used to wash between steps. Afterwards, slides were incubated with the secondary antibody BrightVision poly-HRP-anti-Ms/Rb/Rt IgG (DPVO999HRP, ImmunoLogic) at room temperature for 30 min. Finally, incubation with EnVision™ FLEX DAB Buffered Substrate and EnVision™ FLEX Substrate Buffer (K5207 and SM803; DAKO) was done for 10 min at room temperature prior to dehydration, counterstaining with hematoxylin and finally enclosing with Quick-D mounting medium (7281, Klinipath). Finally, a pathologist evaluated antigen expression as either positive or negative.