Am33034pu n

2 μm FFPE tissue sections of tumor resection specimens were deparaffinized and rehydrated by xylene and sequential decreasing percentages of ethanol. Immunohistochemical staining was performed as described previously.¹² (link) Following epitope retrieval using 10 mM of citric acid monohydrate solution, pH 6.0, in a microwave oven three times for 5 minutes at 560 W, endogenous peroxidase was quenched by 2% hydrogen peroxide solution. After blocking nonspecific binding by incubation of the slides with serum, tissue sections were incubated with the mouse monoclonal antibodies HCA-2 (AM33034PU-N; Acris) and HC-10 (AM33035PU-N; Acris) that recognize a variety of human HLA-A and HLA-B proteins,⁴⁰ (link) both at a dilution of 1:150 at 4°C overnight. Next, secondary antibody biotin coupled horse anti-mouse IgG (1:100 dilution, BA2000; Vector) was applied on the slides and Vectastain Elite ABC kit (Vector) was used to amplify the signal. To develop the slides, liquid DAB and substrate chromogen system (K3468; Dako) was utilized. Hematoxylin/Eosin staining was used to counterstain the slides. The stained samples were visualized using an Olympus BX43 microscope and the cell^D software (version 2.6, Olympus, Duesseldorf, Germany).