Immunohistochemistry

The formalin-fixed paraffin-embedded slides (4μm) were immunostained with primary antibodies against EI24 (H-20, goat polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, dilution in 1:200) as described previously [45] and evaluated by LL and XW. Sections incubated with PBS instead of antibodies were used as the negative control. A total of 200 tumor cells chosen randomly in each section were counted and the percentage of positive tumor cells was evaluated. The immunostaining results were categorized as following: negative (<10% cytoplasmic staining); weakly positive (10%-25%); moderately positive (25%-50%); and highly positive (≥50%). Then negative and weak staining was defined as low expression of EI24, while moderate and strong positivity was defined as high expression of EI24.