Ventana benchmark ultra ihc staining system

After tumor resection, the specimens were macroscopically examined by a pathologist, fixated in 4% buffered paraformaldehyde and sectioned. Preparation for histological examination, including dehydration, paraffin embedding and hematoxylin and eosin (HE) staining were carried out due to standard protocols at the Gerhard-Domagk-Institute, University Hospital Münster. Histological examination was performed by two pathologists (A.K and S.H.).
Immunohistochemistry was done at the Gerhard-Domagk-Institute using the automated Ventana BenchMark ULTRA IHC staining system (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. Concisely, 3 µm sections from paraffin-embedded tissue were deparaffinized and pre-treated with Cell Conditioning 1 solution (CC1, Ventana/Roche, Basel, Switzerland) for 24–64 min at 95–100 °C. Subsequently, incubation with primary antibodies (AR, clone SP107, CellMarque, Rocklin, CA, USA; SV40LT, clone PAb101-412, Invitrogen, Waltham, MA, USA) was implemented for 16–32 min at 36 °C. Visualization of immunoreaction was done via Optiview DAB IHC Detection Kit. Tissue sections were counterstained with hematoxylin and blueing solution (all Ventana/Roche).

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).