Flex rabbit envision system

Immunohistochemistry (IHC) was performed on representative whole formalin fixed, paraffin embedded (FFPE) 5 micron-thick tissue sections. Heat induced epitope retrieval was performed with FLEX TRS Low pH Retrieval buffer (6.10; Dako, Carpinteria, CA) for 20 minutes. After peroxidase and protein blocking, the rabbit polyclonal FOXI1 antibody (1:250; Atlas Antibodies, Stockholm, Sweden) or rabbit polyclonal RHCG antibody (1:4000; LSBio, Seattle, WA) was applied to the sections and incubated at room temperature for 60 minutes. The FLEX + Rabbit EnVision System (Dako, Carpinteria, CA) and DAB chromogen were used for detection. Immunopositivity was defined as brown pigmentation in the membrane/cytoplasm (for RHCG) or nucleus (for FOXI1) of tumor cells. The staining was independently assessed by two pathologists (SLS and RM) in each case for presence and pattern of expression.

Immunohistochemical staining for HER2 overexpression was performed on LSCC and OSCC TMA samples. Briefly, the formalin-fixed paraffin-embedded sections from the TMAs were heated, and underwent peroxidase blocking. A HER2 rabbit monoclonal antibody (SP3; Cell Marque, Rocklin, CA) was applied at a 1:150 dilution. A FLEX + Rabbit EnVision System (Dako, Carpinteria, CA) was used for staining. The TMAs were then counterstained with Harris Hematoxylin. Protein expression was scored by a board-certified pathologist in a blinded fashion, and samples with 3+ or 4+ scoring were counted as positive samples, in accordance with current national guidelines^{21 (link)}. Additionally, staining and scoring for EGFR overexpression was performed in a similar fashion on the TMA samples.