Immunohistochemical Analysis of ETS2 and Phospho-ETS2

Immunohistochemistry was performed as previously described (Bian et al., 2009 (link)). Briefly, 5-μm sections were cut, stained with H&E or processed for immunohistochemistry/immunofluorescence microscopy. For immunoperoxidase staining, paraffin-embedded sections were dehydrated and antigenic epitopes exposed using a 10-mM citrate buffer (pH 6.0) in a pressure cooker. Sections were incubated with the following primary antibodies at 4°C overnight: rabbit anti-ETS2 (1:100; LifeTechnologies PA5-28053) and rabbit anti-phospho-ETS2 (1:100; LifeTechnologies 44-1105G). Primary antibody staining was visualized using peroxidase-conjugated anti-rabbit IgG followed by the DAB substrate kit for peroxidase visualization of secondary antibodies (Vector Laboratories, Burlingame, CA). Tissue microarrays comprising healthy human skin samples, human skin SCCs as well as head and neck SCCs (HNSCC) were obtained from US Biomax, Rockeville, MD: SK241, SK801b, SK811a, SK2081, and HN803a.