54 Chapter 3 Figure 2: Demonstration of the complementarity between D1C2 and its blocking peptide. After incubation of D1C2 with its blocking peptide, the complementarity of these reagents was checked under reducing, native and FFPE conditions by means of E. western blotting (antibody validation cell line panel). F. immunocytochemistry (HT-29). G & H. immunohistochemistry (HT-29 & representative oral SCC). A through D. Corresponding D1C2 immunoreactivities. Negative controls – of the latter immunocyto- & immunohistochemistry experiments – are depicted in the inlays. Considering the made assumptions, the results indicate that SP44, C-12 and C-28 are not specific in the detection of MET protein product and fragments across all examined conditions and are therefore considered to behave nonspecifically. In contrast, D1C2 and CVD13 are specific across all examined conditions. In comparison to one another, D1C2 is slightly more sensitive in the detection of p170MET and p145MET under reducing conditions. Under native and FFPE conditions, D1C2 is more sensitive in the detection of membranous MET and CVD13 is more sensitive in the detection of cytoplasmic MET. Furthermore, the complementarity between D1C2 and the peptide that was used for its generation was confirmed under all investigated conditions. Evaluation of MET immunoreactivity in a cohort of oral and oropharyngeal SCC, a scatter plot based analysis To study MET immunoreactivity in a series of oral and oropharyngeal SSC, a TMA was stained with the antibody that is most sensitive in the detection of membranous MET (i.e., D1C2). With cores taken from the center and periphery, the design of the TMA allows to explore the behavior of MET immunoreactivity across cancer surfaces. Further details concerning the design of the TMA are described in the Materials and Methods section, paragraph patient material.
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