Immunohistochemistry

Tissues were fixed with paraformaldehyde, embedded with paraffin, then cut into sections of 5 µm thickness for immunohistochemistry (IHC) analysis mainly according to literature studies (20). The first antibody against IQGAP1 (ab133490, Abcam), CDC (ab187643, Abcam) was respectively used at a dilution of 1:100, 1:400. The second antibody was a biotinylated IgG for 40 min incubate at 37°C. Tissue slices were visualized by the 3, 3′-diaminobenzidine solution, and cellular nuclei were slightly counterstained with hematoxylin. Substitution of the primary antibody with phosphate-buffered saline (PBS) was taken as a control for IHC. According to general evaluation standards (21), the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). The extent of staining was monitored based on the percentage of positive tumor cells: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%). The final score of 0 was defined as a negative expression (−); scores of 1–3 were accepted as a low/weak expression (+), and scores over 3 were defined as a high/strong expression (++). The intensity and percentage of positive cells were evaluated at least in five separate fields at a 400-fold magnification. The IHC scores of one tissue sample was respectively determined by two pathologists, and the final score for a tissue sample was calculated from the average value of the two sets of total scores. P<0.05 was considered statistically significant.