Clone qbend 10

IHC was performed on 3 µm sections from paraffin-embedded tissue using the automated Ventana BenchMark ULTRA IHC staining system (Roche). Tissue sections were deparaffinized and pre-treated with Cell Conditioning 1 solution (CC1, Ventana/Roche) for 24–64 minutes at 95–100 °C. The primary antibodies were then incubated for 16–32 minutes at 36 °C. Immunoreactions were visualized via the Optiview DAB IHC detection kit (ref.no. 760–700, Ventana/Roche). Tissue sections were counterstained with hematoxylin (ref.no. 790-2208, Ventana/Roche) and bluing solution (ref.no. 760-2037, Ventana/Roche). The mouse polyclonal β-catenin antibody (ref.no. CMC 22421040, clone 14, CellMarque) was applied. Only nuclear localization of β-catenin was considered positive for the purpose of the study, and the percentage of stained tumor cells was estimated in 10 high-power microscopic fields (400x magnification). DTF cases with ≥10% immunoreactive cells were regarded as positive. Following antibodies were additionally applied to exclude differential diagnoses: CD34 (ref.no. 790-2927, clone QBEnd10, Ventana/Roche), DOG1 (ref.no. CMC 24431050, clone SP31, CellMarque), CDK4 (ref.no. AHZ0202, DCS-31, Invitrogen/Thermo Fisher Scientific) and MDM2 (ref.no.33-7100, IF2, Invitrogen/Thermo Fisher Scientific).