Immunohistochemistry

Immunohistochemistry staining was performed at the department of pathology in The Second Affiliated Hospital of Soochow University. The tissue blocks were cut at sections in 5 μm thick. After baked for 60 min, paraffin sections were deparaffinized in xylene and ethanol. Deparaffinized sections were then placed in 0.01 mol/L sodium citrate buffer, pH 6.0. The buffer was brought to a boiling temperature and timed for 30 min. Sections were incubated with 3% H2O2 for 10 min. After blocked with 10% goat serum for 15 min, the sections were further incubated with primary antibodies (4°C, overnight). The sections were then washed three times with PBS and incubated with secondary antibody for 20 min. The sections were stained by DAB Substrate kit and counterstained with hematoxylin. All stains were manually evaluated by one experienced pathologist who was blinded to sample identify. The German Immunoreactive Score was established basing on multiply the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) by extent of immunoreactive cells (0% = 0; 1~10% = 1; 11~50% = 2; 51~80% = 3; 81~100% =4). The interpretation score was defined as follows: 0~1 (0, negative), 2~4 (1+, weakly positive), 6~8 (2+, moderately positive), 9~12 (3+, strongly positive).