P53 monoclonal antibody

Standard ABC peroxidase techniques were used for immunohistochemistry performed on 4 μ sections of formalin-fixed and paraffin-embedded tissue. Antigen retrieval in heated citrate buffer at pH 6.0 was applied for all antibodies. The Ki67 monoclonal-antibody (1:100), Rb monoclonal-antibody (1:400), p53 monoclonal-antibody (1:500), Chromogranin-A polyclonal-antibody (1:8000), and synaptophysin (1:500) were obtained from Dako (Carpentaria, CA). The SMAD4 monoclonal-antibody (1:800) was acquired from Santa Cruz Bio (Santa Cruz, CA). The ATRX polyclonal-antibody (1:500) and DAXX (1:100) polyclonal-antibody were obtained from Sigma-Aldrich Corporation (St. Louis, MO). Immunohistochemistry was performed on BenchMark XT automated equipment (Ventana Medical System Inc., Tucson, AZ). Positive control tissue was stained in parallel with each study case. The Ki67 immunoreactivity was expressed as the percentage of tumor cells with nuclear staining, which was based upon digital counting >2,000 tumor cells in regions with the highest labeling recognizable on scanning magnification. p53 immunoreactivity with strong staining intensity in >25% tumor cells was regarded as abnormal (positive), and complete loss of SMAD4, DAXX, ATRX, and Rb protein expression (negative), in the presence of positive staining in non-neoplastic cells, was regarded as abnormal.

Standard ABC peroxidase techniques were used for immunohistochemistry performed on 4 μ paraffin sections of formalin-fixed, paraffin-embedded tissue. Antigen retrieval in heated citrate buffer at pH 6.0 was applied for all antibodies. The Ki67 monoclonalantibody (1:100), Rb monoclonal-antibody (1:400), p53 monoclonal-antibody (1:500) Chromogranin-A polyclonal-antibody (1:8000), and synaptophysin (1:500) were obtained from Dako (Carpinteria, CA). Immunohistochemistry was performed on BenchMark XT automated equipment (Ventana Medical System Inc., Tucson, AZ). Positive control tissue was stained in parallel with all the study cases. Ki67 immunoreactivity was expressed as the percentage of tumor cells with nuclear staining, based on counting >2000 cells in the regions with the highest labeling recognizable on scanning magnification. The Ki67 indices for the regions with G1/G2 morphology and high grade morphology were recorded separately. p53 immunoreactivity, with strong intensity in >25% tumor cells, was regarded as abnormal, and complete loss of Rb protein expression was regarded as abnormal for this marker.