Anti ki67 clone mib 1

Immunohistochemical (IHC) staining from human tissue microarray or mouse xenograft samples was performed on 3-μm-thick sections from formalin-fixed paraffin-embedded samples and dried in a 37 °C oven overnight. Hematoxylin/eosin (H&E) staining was performed with H&E Leica Kit Infinity for a full automated autostainer (Leica ST5020). IHC staining for Ki67 and MGP antibodies was performed using Bond III IHC autostainer for full Automated Immunohistochemistry (Leica biosystems). Antigen was unmasked with Tris-EDTA pH 9 (Bond Epitop Retrival Solution 2 Leica, cat# AR9640). Both mouse monoclonal anti-Ki67 CloneMIB-1 (Dako, cat# M7240) and rabbit polyclonal antibody MGP (ProteinTech, cat# 10734-1-AP) were used at 1:200. The antibodies were diluted with Bond Primary Antibody Diluent AR9352 Leica. BOND IHC Polymer Detection Kit (cat# DS9800) was used to stain both antibody with DAB Cromogen. IHC samples were counterstained using Hematoxylin solution (Leica, cat# RE7107-CE). Pictures of stained sections were acquired with the Aperio ScanScope XT instrument. Ki67 and MGP staining were analyzed and scored by a board-certified pathologist (GB).

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).

Hematoxylin and eosin (H&E) and IHC stainings were performed at the Mouse Pathology Facility of Lausanne University (Epalinges, Switzerland). IHC labeling was performed using the mouse monoclonal anti-human vimentin Ab (clone RV202, GeneTex, CA, USA), anti-Ki67 (clone MIB-1, Dako, Denmark), and anti-cleaved caspase-3 (ASP175)(#9661, Cell Signaling Technology Inc.) on 4 μm-thick paraffin-embedded mouse tissue sections.