Antibody specific for c myc

Formalin-fixed, paraffin-embedded breast tissue was dewaxed, rehydrated and antigen retrieval was achieved by incubation in 10 mm Tris, 1 mm EDTA buffer (pH 9) for 30 s at 125 °C in a Menapath Antigen Access Retrieval Unit (A Menarini Diagnostics, Wokingham, UK). The tissue was incubated in 3% (v/v) hydrogen peroxide solution for 10 min at room temperature and then washed in 0.02 m Tris-HCl, 0.14 m NaCl and 0.1% (v/v) Tween-20. It was also incubated at room temperature for 60 min with an antibody specific for c-MYC (Epitomics, Burlingame, CA, USA; cat. no.:1472-1) in Tris-buffered saline (pH 7.6) and then with Menapath HRP Polymer (A Menarini Diagnostics) for 30 min. Sections were visualised with 3,3′ diaminobezidine and were counterstained with Gills II haematoxylin. c-MYC expression was quantified by the analysis of 100 malignant epithelial cells in each of five fields. The intensity of the immunoreaction was scored from 0 to 3 (Supplementary Figure 4) by two independent researchers. A histoscore was calculated for each case from the sum of the scores for 500 cells.