Expression of CDCP1 and PDGFRβ was analyzed by IHC in consecutive 2-μm formalin-fixed, paraffin-embedded (FFPE) tumor sections, using rabbit polyclonal anti-CDCP1 (1:50) (PA5–17245, Thermo Fisher Scientific) and rabbit anti-human PDGFRβ (1:200) (Y92, Abcam), respectively. Antigen retrieval was performed by heating the sections for 5 min at 96 °C in 10 mM citrate buffer, pH 6.0. Staining was visualized using streptavidin-biotin-peroxidase (Dako, Agilent Technology, Santa Clara, CA) and 3,3′-diaminobenzidine (DAB; brown signal) (Dako), and the sections were counterstained with hematoxylin. Images were acquired by ECLIPSE TE2000-S inverted microscope (Nikon Instruments, Melville, NY) at 20X and 40X magnification. The reactivity of anti-CDCP1 and anti-PDGFRβ was considered to be positive per Turdo 2016 and D’Ippolito 2016 [3 (link), 25 (link)]. Specifically, based on the intensity of PDGFRβ staining in neoplastic cells, we assigned tumors a score of 0 (absence of signal) or 1 (weak to strong cytoplasmic signal and membrane signal). Reactivity of polyclonal anti-CDCP1 was defined as positive when ≥10% of tumor cells showed membrane staining.
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