Tissue microarray (TMA) slides were generously provided by Prof. Zhang Zhigang (Shanghai Cancer Institute, China). After deparaffinization and hydration in Milli-Q water, antigens were retrieved by boiling TMA slides in citrate buffer for 20 min. Then, endogenous peroxidase activity and nonspecific binding activity were blocked before incubation with the primary antibody at the recommended dilution (4 °C, overnight) in a wet box. Finally, immunoreactivity was visualized using the Mouse and Rabbit specific HRP/DAB (ABC) Detection IHC Kit (Abcam, Cambridge, MA, USA) in accordance with the manufacturer's instructions. Nuclei were counterstained with hematoxylin (Sigma-Aldrich). Images were taken by a NanoZoomer S60 slide scanner (Hamamatsu Photonics, Shizuoka, Japan) at a magnification of 20×. The expression levels of corresponding targets were determined by quantitative analysis. IHC scores were graded according to the percentage of stained cells as previously described 38 (link). Briefly, the scoring was assigned as follows: the percentage scores: 0, <5% of positively stained cells; 1, 5-50%; 2, 51-100%; the intensity scores: 0, absent or faint; 1, weak; 2, moderate; 3, strong.
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