Horseradish peroxidase detection system

Tissue sections were deparaffinized in xylene and rehydrated with ethanol, and blocking of endogenous peroxidase activity with 3% hydrogen peroxide was followed by microwaving in 0.01 M sodium citrate buffer for antigen retrieval, after which the slides were preincubated in 10% normal goat serum for 1 h, followed by incubation overnight at 4 °C with the indicated antibodies. Afterwards, the expression of the indicated proteins was detected by a horseradish peroxidase detection system according to the manufacturer’s instructions (DAKO, Glostrup, Denmark).
The immunohistochemically stained tissue sections were scored separately by three pathologists blinded to the clinicopathological parameters. The staining intensity was scored as follows: 0, no staining (negative); 1, yellow (weak); 2, brownish yellow (medium); and 3, brown (strong). In the same tissue, multiple high-power fields with different staining intensities were viewed, and the percentage of cholangiocarcinoma-positive cells was calculated separately and then taken as the average. Scores for staining intensity and percentage of positive cells were then multiplied to generate the immunoreactivity score for each case. Samples with a final staining score of ≤ 1 were considered to have low expression, and those with a score of >1 were considered to have high expression.

The tissues after formalin-fixed and paraffin-embedded were cut into 4-μm sections. The specimens were deparaffinized in xylene and rehydrated using a series of graded alcohols after being dried at 62 °C for 2 h. The slides were then treated with 3% hydrogen peroxide in methanol for 15 min. To exhaust endogenous peroxidase activity, the antigen was retrieved in 0.01 M sodium cirate buffer (pH 6.0) using a microwave oven. After 1 h of preincubation in 10% goat serum, the specimens were incubated with a primary Ab at 4 °C overnight. The slides were treated with a horseradish peroxidase detection system (DAKO, Glostrup, Denmark) according to the manufacturer’s instruction. Two independent individuals evaluated the slides. The intensity of immunostaining was taken into consideration when analyzing the data.