Immunohistochemistry controls and antibody specificity. (a) Immunohistochemical staining with control IgG for tumors after the first cycle of epirubicin plus cyclophosphamide (EC) treatment (upper panel). Lower panel shows Rad51 staining for morphologically diagnosed non-cancerous breast tissues (left upper part) and tumor (right lower part) after the first cycle of EC treatment. Although non-cancerous breast cells also expressed nuclear foci formation the number and intensity was significantly lower than that in tumor cells. (b and c) DNA damage-induced nuclear foci formations detected by antibodies used in the study. HeLa cells were either untreated (-), treated with 5 Gy ionizing radiation (IR) or 0.2 μg/ml epirubicin (Epi), incubated for three hours and fixed. Cells were then subjected either to immunofluorescent analyses with the indicated primary antibodies and (b) FITC- (green) or Rhodamine- (red) conjugated secondary antibodies, or (c) to the same protocol as that used in the tissue stain. For immunofluorescent analyses the nucleus was counterstained with DAPI. Ub, conjugated ubiquitin.