Immunodetection of Cancer Antigens

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)}.