PMC:4385186 / 19642-20953 JSONTXT

{"project":"2_test","denotations":[{"id":"25839328-23726842-2052573","span":{"begin":59,"end":61},"obj":"23726842"}],"text":"Immunohistochemistry was performed as previously described.17 In brief, sections were incubated with special antibody at an ideal dilution for 14 hr at 4°C and then detected with PV8000 (Zhongshan) and the DAB detection kit (Maixin), producing a dark-brown precipitate. Slides were counterstained with hematoxylin. All images were captured at 100×. The nuclear amount of the protein of interest was analyzed with Aperio Nuclear v.9 software, and the cytoplasmic protein amount was quantified with Aperio Cytoplasma 2.0 software. Statistical analyses were performed with GraphPad Prism 5.0. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The immunoreactive score (IRS) was determined by the product of the extent score and the intensity score. IRS values ranged from 0 to 9, which were graded as follows: 0 (negative), 1–3 (weak), 4–6 (moderate), and 7–9 (strong). The median IRS was chosen to define the individuals, and the ratio of tumor to matched normal tissue (TIRS/NIRS) was used to compare the protein of interest with significantly high amounts (TIRS/NIRS \u003e 2), high amounts (1 \u003c TIRS/NIRS \u003c 2), low amounts (TIRS/NIRS \u003c 1), or no change (TIRS/NIRS = 1) in tumor tissue to that in matched normal tissue. All antibodies used in this study are shown in Table S5."}