Pan cytokeratin clone mnf116

Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned (4 µm) and forwarded to either hematoxylin and eosin (HE) or immunohistochemical staining using antibodies against brachyury (clone H-210; Santa Cruz, Santa Cruz, USA), pan-cytokeratin (clone MNF116, Dako), epithelial membrane antigen (EMA; clone E29, Dako) and S100 (polyclonal; Dako, Glostrup, Denmark). Tissue sections were subjected to antigen retrieval in a tris-borate/EDTA buffer (CC1, Ventana, Roche, Vienna, Austria) before incubating with clone H-210 (Santa Cruz, dilution 1∶50) for brachyury immunohistochemistry. Antibody detection was performed on a Ventana Immunostainer using the ultraView Universal DAB Detection Kit (Roche) according to the manufacturer’s recommendations.

FFPE blocks from ONB tissue were generated at the time of primary surgery from radiotherapy (RT)-naïve patients. All cases were reviewed by two expert head and neck pathologists (S.B. and L.A.) and the diagnosis confirmed using hematoxylin and eosin staining. In case of disagreement about grading between the two pathologists, a final grade was reached by consensus.
All immunohistochemical analyses were performed on serial 5 µm sections of a selected FFPE representative tumor block. The markers used for diagnostic purposes were chromogranin A (clone LH2H10, DB-Biosystem, Franklin Lakes, NJ, USA), synaptophysin (clone DAK-SYNAP, Dako, Glostrup, Denmark), S100 protein (polyclonal, Leica, Wetzlar, Germany), pan-cytokeratin (clone MNF116, Dako, Glostrup, Denmark) and Ki-67 proliferation index (clone 30-9, Ventana, AZ, USA). Immunohistochemical analysis was performed on the entire slide of the selected block and the percentage of positive neoplastic cells was determined. Quantification of the Ki-67 index was done by counting the positive neoplastic cells among all tumor cells present in the area of interest (hotspot area) and by determining the percentage of positive neoplastic cells in the entire tumor area.