Anti emmprin

Four-micrometer thick paraffin-embedded sections were deparaffinized and heated in a microwave oven (700 watts) for 10 min to expose antigens in a 10 mM Na-citrate buffer, followed by blocking in 3% hydrogen peroxide solution. The sections were incubated with anti-emmprin (mouse monoclonal, R&D, 1:200) and CD73 (rabbit polyclonal, Abcam, 1:100) antibodies at 4 °C overnight. The exposed antigen was detected using EnVision reagent conjugated horseradish peroxidase (Agilent, Santa Clara, CA). The reaction was identified with DAB and counterstained with Mayer’s hematoxylin. The staining results were evaluated semi-quantitatively by two independent observers. Immunostaining was considered negative if the percentage of stained tumor cells or stromal cells was < 10%. In specimens considered positive, staining of the cells was quantified on a scale of 1–4 based on the percentage of positive cells. The scale was structured as follows: 1+, 10–25% of cells positive; 2+, 25–50% of cells positive; 3+, 50–75% of the cells positive; 4+, > 75% of the cells positive. CD73-close and CD73-distant staining score were means of three high power fields of view. CD73-close included stromal cells positioned close to the tumor cells, and CD73-distant were those most distant from the tumor cells in the same slides.