Streptavidin biotin peroxidase

Expression of CDCP1 was analyzed by IHC on consecutive 2-μm formalin-fixed, paraffin-embedded (FFPE) tumor sections, using rabbit polyclonal anti-CDCP1 (1:50) (PA5-17245, Thermo Fisher Scientific), which is directed against the C-terminus of CDCP1, after antigen retrieval, which as performed by heating the sections for 5 min at 96°C in 10 mM citrate buffer, pH 6.0. Immunoreactions were visualized using streptavidin-biotin-peroxidase (Dako, Agilent Technology, Santa Clara, CA), 3,3′-diaminobenzidine (DAB; brown signal) (Dako) as the chromogen, and the sections were counterstained with hematoxylin. Images were acquired on an ECLIPSE TE2000-S inverted microscope (Nikon Instruments, Melville, NY) at 20× and 40× magnification.
The reactivity of anti-CDCP1 in the TNBC specimens was considered to be positive when ≥ 10% of tumor cells showed membrane staining. This cutoff was chosen, based on distribution analysis of the percentage of CDCP1-positive cells by IHC in each tumor section. No tumors had < 10% CDCP1-positive cells in our series, and cases with different percentages of CDCP1-positive cells were likewise distributed in a 10–100% interval. By explorative Kaplan-Meyer analysis of DFS in our cases—stratified as negative, ≥ 10% and < 50%, or ≥ 50% for CDCP1 expression, both CDCP1-positive groups had a worse and superimposable DFS compared with CDCP1-negative cases (data not shown).

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.

The paraffin-embedded tissue sections were deparaffinized using xylene and dehydrated with graded alcohol and followed by PBS wash. The slides were incubated in citrate buffer (pH 6.0) for antigen retrieval using wet autoclaving method. The sections were allowed to cool to room temperature. The sections were treated for 25 min with 3% H₂O₂ in 1X TBST to inhibit endogenous peroxidase activity. After blocking in 1% BSA-PBS for 45 min, the sections were incubated overnight in primary antibody prepared at a dilution appropriate for each of the proteins in blocking buffer at 4 °C overnight. The slides were washed with TBS and then incubated with biotin-labeled secondary antibody followed by streptavidin-biotin-peroxidase (Dako, Carpinteria, CA, USA) for 30 min each at room temperature. The immunoprecipitate was visualized by treating with 3,3 -diaminobenzidine and counterstaining with hematoxylin. The tissues were then photographed using an Inverted Fluorescence Microscope (Leica Microsystem Vertrieb GmbH, Wetzler, Germany) attached with digital camera DFC295.