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.