Immunohistochemistry (IHC) staining was performed as previously described (18 (link), 19 (link)). In brief, 4-µm sections from representative breast cancer tumor tissue were cut from formalin-fixed paraffin-embedded specimens and underwent deparaffinization, rehydration, endogenous peroxidase blocking, and antigen retrieval. The following primary antibodies were used: PTPRO (Cat# sc-365654, Santa Cruz, CA, USA), and CD8 (Cat# ab101500, Abcam, Cambridge, UK). Furthermore, the primary antibodies were incubated at 4°C overnight. Then, the sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, followed by color development with 3,3′-diaminobenzidine (DAB) substrate kit (DAKO, Glostrup, Denmark). The nuclei were counterstained with hematoxylin.
The percentage of PTPRO expression in the tumor cells was scored using the following scales: 0, negative; 1, ≤10%; 2, 11%–50%; 3, 51%–75%; and 4, >75%. The intensity of staining was scored using the following scales: 1, weak staining; 2, moderate staining; and 3, strong staining. The percentage (P) and intensity (I) of the cytoplasm or membrane expression were multiplied to generate a numerical score (S = P*I), which was modified from previous studies (19 (link)).
The percentage of PTPRO expression in the tumor cells was scored using the following scales: 0, negative; 1, ≤10%; 2, 11%–50%; 3, 51%–75%; and 4, >75%. The intensity of staining was scored using the following scales: 1, weak staining; 2, moderate staining; and 3, strong staining. The percentage (P) and intensity (I) of the cytoplasm or membrane expression were multiplied to generate a numerical score (S = P*I), which was modified from previous studies (19 (link)).