Immunohistochemistry analysis on NB-tissue

In order to characterize the TP53 mutational status and Ki67 proliferation index, which are associated with NB prognosis, we performed an immunohistochemistry analysis on the seven NB patients enrolled in the study. Tissue samples were fixed in 10% neutral buffered formalin for at least 24 h, washed in water for 30 min and placed in ethanol 70% before the automated processing (Leica ASP300 S system). Then, samples were included in High Melt Paraffin (Surgipath Paraplast) and 5 μm thick sections were obtained by using the microtome. For immunohistochemistry analysis of Ki67 and TP53 DO-7 staining, the slides were dewaxed and then subjected to automated retrieval using DAKO PT link (Agilent) in 0.1 M citrate buffer pH 6.0 or pH 9.0 according to the manufacturer’s instructions. Upon overnight exposure at 4 °C to the following primary antibodies, Ki67 (mouse IgG1; Dako Cytomation) and TP53 DO-7 (mouse IgG2b; Novocastra Leica) tissue samples were incubated with biotinylated anti-mouse by using the LSAB 2 Kit (Dako Cytomation or Vectastain kit; Vector) following the manufacturer’s instructions. Staining was detected using 3-amino-9- ethylcarbazole (AEC) chromogen. Nuclei were counter-stained with hematoxylin (Sigma).