Ventana benchmark ultra ihc ish system

AsPC-1 and Capan-2 cancer cell pellets and human pancreatic cancer tissue microarrays (PA483e, Insight Biotechnology/US Biomax) were used to determine whether novel monoclonal antibodies immunodetect the target antigens in formalin-fixed paraffin-embedded tissue sections using the VENTANA BenchMark ULTRA IHC/ISH System (Roche, UK). Tissue sections were deparaffinised and rehydrated through a series of alcohols followed by heat induced antigen retrieval with standard CC1 (Tris-EDTA buffer pH 7.8) at 95 °C for 36 min and incubation for 1 h with either mAb KU44.22B (15 µg/ml) or mAb KU44.13A (10 µg/ml). An anti-mouse IgG detection system (UltraView Universal DAB Detection Kit, Ventana) was used for amplification and primary antibody detection. The slides were then counterstained with haematoxylin II for 8 min followed by Bluing reagent. The slides were then washed, dehydrated, cleared, mounted in DPX mounting medium (VWR) and coverslipped as described previously^{60 (link)}.

EBER in situ hybridization was performed using a commercially available kit. Briefly, formalin-fixed, paraffin-embedded sections were hybridized with the Inform EBER probe (#05278660001, Roche Diagnostics) and the VENTANA ISH iView Blue Detection Kit was used for visualization (#05278511001, Roche Diagnostics). The staining procedures were performed in a Ventana BenchMark ULTRA IHC/ISH System (Roche Diagnostics) according to guidelines from the supplier.

Immunohistochemical reactions were performed on 4 μm thick FFPE preparations. Expression of TFAP2B protein was detected using rabbit anti-AP-2β antibody (sc-8976, dilution 1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Antigen retrieval was performed at pH 6.0 for 30 min. at 99 °C. Expression of ALK protein was detected using antibody clone D5F3 #3633 (Cell Signalling, Denver, MA, USA) at dilution 1:250 and antigen retrieval was performed using EnVision FLEX HIGH pH solution (DAKO) for 30 min. at 99 °C. Expression of OLIG2 was detected using antibody EP112 (Cell Marque) at concentration 1.92 μl/ml and antigen retrieval was performed in CC1 buffer using Ventana BenchMark ULTRA IHC/ISH system (Roche).
TFAP2B and ALK scoring were as the following: negative for < 10%, intermediate for 10–50% and positive for > 50–100% of tumour cells. OLIG2 was considered positive when > 10% of tumour cells displayed positive nuclear reaction.
Whole preparations were scanned in Hamamatsu NanoZoomer 2.0 RS scanner at original magnification 40x.