Rb if8

IHC was performed using the Dako Envision system (Dako, Carpinteria, CA), as described previously26 (link). Primary antibodies against human p53 (DO-1, diluted 1:100), Rb (IF 8, diluted 1:100), p-Rb (Ser 807, diluted 1:100), and Gankyrin (H-231, diluted 1:400) were purchased from Santa Cruz Biotechnology, INC., Santa Cruz, CA. Appropriate positive and negative controls were used.
Nuclear or cytoplasmic staining was considered specific Gankyrin immunoreactivity. For p53, Rb, and p-Rb, only nuclear labeling was regarded as positivity. The status of immunostaining was evaluated in a semiquantitative manner by considering both the intensity and percentage of positive cells to generate a histochemical score (H-score), as described previously26 (link). For Gankyrin staining, each case was assigned two H-scores, one each for nuclear and cytoplasmic staining. A final total cellular H-score for Gankyrin staining was determined as follows: total cellular score = H-score of nuclear staining + H-score of cytoplasmic staining. A minimum of 100 cells were evaluated to calculate the H-score. All sections were independently evaluated by two experienced pathologists who were blinded to the clinical and pathological data. When the opinions of the two pathologists differed, an agreement was reached by careful discussion.