48 Chapter 3 biotin to reduce non-specific background staining. Instead of 90 seconds, horseradish peroxidase activity was visualized in 2x 5 minutes. The used primary antibody titers are summarized in and the sections were counterstained with haematoxylin before mounting them with a coverslip. Evaluation of MET immunocyto & immunohistochemical staining Cells included in the antibody validation panel and whole tissue sections of oral SCC D1C2, SP44, CVD13, C-12 and C-28. Staining intensities were evaluated for nuclear, cytoplasmic and membranous immunoreactivity on a scale from 0 to 3. For nuclear and cytoplasmic immunoreactivity this means: 0 = no staining, 1 = weak staining, 2 = moderate staining and 3 = strong staining. For membranous immunoreactivity this means: 0 = no staining, 1 = weak complete or incomplete membranous staining, 2 = moderate complete membranous staining and 3= strong complete membranous staining. Representative images of nuclear, cytoplasmic and membranous staining intensities are depicted in Supplementary Figure S1D. The final immunoreactivity score was defined as the maximum observed staining intensity involving at least 10% of the cancer cells. Scores were given separately for the nucleus, cytoplasm and membrane. The cell lines cultured on chamber slides were evaluated in areas with equal cell density and similar morphology to assure comparability. In case of the whole tissue sections, well-differentiated cancer cells that show no nuclei were omitted during scoring. Two observers (SK and MDH) simultaneously assessed the immunoreactivity scores. TMA representing a cohort of oral and oropharyngeal SCC D1C2. Evaluation of MET staining intensities was restricted to membranous immunoreactivity as defined above. Cancer cells showing no (0) to weak (1) membranous immunoreactivity were assessed as negative for MET, while cancer cells showing moderate (2) to strong (3) membranous immunoreactivity were assessed as positive for MET. Moreover, seemingly suboptimal fixed cores and well-differentiated cancer cells that show no nuclei were omitted during scoring.
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