IHC staining was performed at RT on an automatic staining workstation (Dako Autostainer Plus, Glostrup, Denmark) using anti-SDCBP (ab133267, Abcam, Cambridge, UK), anti-p21 (Clone 4D10; Leica Biosystems NCL-L-WAF-1, Barcelona, Spain) at a 1:10 dilution, anti-E-cadherin (BD Biosciences, CA, USA) at a 1:4000 dilution, anti-Src (active) Clone 28 (#AHO0051, Thermo Fisher Scientific, Waltham, MA, USA) at a 1:300 dilution, anti-Ki67 (clone MIB-1, Dako, Glostrup, Denmark) for 30 min, and anti-Nanog and anti-Notch antibodies, as described. [21 (link)] Counterstaining with Hematoxylin and eosin was the final step. For the Ki-67, p21, and Notch1 proteins, nuclear staining was evaluated and dichotomized as a negative expression (0–10% stained cells) versus positive expression (>10% stained tumor cells). E-cadherin, Src, Nanog, and Notch1 were scored as described [14 (link),21 (link)]. Validation of SDCBP was performed at the nuclear, membrane, and cytoplasmic levels in each patient according to the immunoreactive score (IRS) by multiplying the quantity and staining intensity scores (Table S1 ).
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