Ace substrate

The tumors were fixed in 4% paraformaldehyde for 24 h (Sigma-Aldrich, St. Louis, MO, USA), washed with PBS, dehydrated in increasing alcohol concentrations and embedded in paraffin blocks. Sections were deparaffinized and rehydrated, treated with proteinase K (20 μg/ml) for 15 min and washed in PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 min. Fragmented nuclear DNA, associated with apoptosis in histological sections, was labeled in situ with digoxigenin-deoxyuridine (dUTP), introduced by terminal deoxynucleotidyl transferase (TdT), using ApopTag^® peroxidase in situ apoptosis detection kit according to manufacturer’s instructions (Intergen, Oxford, England, UK). The reaction was terminated using the ApopTag^® stop buffer followed by anti-digoxigenin-peroxidase application and the labeled nuclei were detected with ACE substrate as the chromogen (Sigma-Aldrich, St. Louis, MO, USA). MVD were labeled using anti-CD34 antibody (Abcam, ab81289, USA). The slides were examined using a Zeiss microscope, and images were processed using ZEN digital imaging software.

The tumors were fixed in 4% paraformaldehyde for 24 h (Sigma-Aldrich, St. Louis, MO, USA), washed with PBS, dehydrated in increasing alcohol concentrations and embedded in paraffin blocks. Sections were deparaffinized and rehydrated, treated with proteinase K (20 μg/ml) for 15 min and washed in PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 min. Fragmented nuclear DNA associated with apoptosis in histological sections was labeled in situ with digoxigenin-deoxyuridine (dUTP), introduced by terminal deoxynucleotidyl transferase (TdT), using ApopTag® peroxidase in situ apoptosis detection kit according to manufacturer's instructions (Intergen, Oxford, England, UK). The reaction was terminated using the ApopTag® stop buffer followed by anti-digoxigenin-peroxidase application and the labeled nuclei were detected with ACE substrate as the chromogen (Sigma-Aldrich, St. Louis, MO, USA). The slides were examined using an Olympus BX-43 microscope, and images were processed using cellSens entry digital imaging software.