Image pro plus 6.0 analytic

After routine deparaffinization and hydration, tissue sections were treated with 0.01 M citrate buffer (pH 6.0) for antigen retrieval. Following 3% hydrogen peroxide incubation and 10% bovine serum albumin deprivation, slides were incubated with primary antibodies of c-Fos (1:100, Cell Signaling), Wnt2 (1:100, Abcam), and Fzd9 (1:100, Abcam) at 4°C overnight. Primary antibodies were recognized by the biotinylated secondary antibody and visualized by Vectastain avidin–biotin complex peroxidase system (Zsbio, China) and 3,3′-diaminobenzidine kit (DAB, Zsbio, China). The degrees of immunoreactivity were evaluated in accordance with our previous report[14 (link)]. We performed staining by only adding phosphate-buffered saline (PBS) instead of using primary antibodies to the sections as negative controls. The sections were photographed using an optical microscope (Olympus, BX53F, Japan) and then analyzed using the Image-Pro Plus 6.0 analytic system.

Immunohistochemistry staining was carried out on 4 μm paraffin sections as previously reported (Liu et al., 2014 (link)). After deparaffinization, rehydration, and heat-induced epitope retrieval, the slides were washed with PBS three times and then incubated with primary antibodies, including a rabbit polyclonal antibody to DDX5 (1:200, Abcam), a rabbit polyclonal antibody to Cyclin E1 (1:80, Anbo), and a rabbit polyclonal antibody to TCF12 (1:100, Millipore), overnight at 4°C. After that, the sections were incubated with secondary antibody for 1 h at room temperature. The secondary antibody is horseradish peroxidase (HRP)-labeled. Colored reactions were developed using diaminobenzidine as chromogenic substrate, visualized using an Olympus BX53F microscope, and analyzed using the system of Image-Pro Plus 6.0 analytic. The degrees of immunoreactivity were evaluated based on the final immunoreactivity score (IRS) by multiplying the percentage of positive tumor cells and the intensity score of staining. An IRS value > 5 was considered as high expression and an IRS value < 5 as low expression (Liu et al., 2014 (link)).