Immunohistochemical assay
Initially, pathological specimens were deparaffinized with dimethyl benzene and rehydrated through an ethanol gradient (100%, 95%, 90%, 80%, and 70%) into water. After washing with PBS (phosphate-buffered saline three times, slides were boiled in antigen retrieval buffer, 0.01 M sodium citrate-hydrochloric acid (pH=6.0), for 30 min in a microwave oven. After endogenous peroxidase activity was quenched with 3% H2O2 after three PBS washes, nonspecific antibody binding was blocked by incubating the slides with 10% normal goat nonimmune serum. The sections were then incubated at 4°C overnight with the rabbit polyclonal RAGE antibody (Santa-Cruz, CA, USA) at a 1:400 dilution and subsequently incubated with horseradish peroxidase (HRP) (ChemMateTM DAKO EnVisionTM Detection Kit) at room temperature for 30 min. After washing in PBS, the sections were then developed using 3,3-V-diaminobenzidine (Sigma), washed in running tap water, and lightly counterstained with hematoxylin before dehydration and coverslip mounting. Negative control experiments were conducted by replacing the primary antibody with PBS.
The RAGE detection was arbitrarily determined as to extent of immunoreactivity: 0, <5%; 1, 5%-10%; 2, 10%-50%; 3, 50%-75%; 4, >75%. Staining intensity was scored as 0, negative; 1, weak; 2, moderate; 3, strong (figure 1). For each case, the total immunohistostaining score, also known as the staining index (SI) was calculated by multiplying the percentage of positive cells with the staining intensity score, yielding a value between 0 and 12. For statistical analysis, staining intensity scores 0 and 1 defined as negative expression (–), 2 to 12 scores defined as positive expression (+)28.