Histology and immunohistochemistry (IHC) of the tumor tissue sections

Paraffin-embedded tissue sections (5 μm) were de-paraffinized, rehydrated with graded alcohol and PBS, and used for histological staining (H&E) and IHC analyses [22–26]. A tissue section of SC tumors derived from the human prostate cancer LNCaP-expressing clone (LNS239) was used as a positive external control for IHC staining [22]. 1/200 to 1/300 dilution of the chicken anti-huMETCAM/MUC18 IGY antibody was used as the primary antibody and 1/250 dilution of the biotinylated rabbit anti-chicken IGY antibodies (G2891, Promega) as the secondary antibody [22–26]. A streptavidin-conjugated horseradish peroxidase complex (Dako LSAAB-2 system) and diaminobenzidine were used for color development. Hematoxylin was used as the counter staining. Negative controls had the primary antibody replaced by non-fat milk or control chicken IGY.