Immunohistochemical Analysis of Tumor Tissues

Immunohistochemistry was performed as published [8 (link)]. Briefly, tumor tissues were dissected, fixed and processed in paraffin wax. Tissue sections on glass slides were pre-treated in the Lab Vision PT Modul (Thermo Scientific, Munich, Germany) and in PT Modul Buffer (pH 6) (TA-250-PM, Medac, Hamburg, Germany). Endogenous peroxidases were blocked by incubation in peroxidase blocking buffer (TA-125-HP, Medac) for 10 min. Primary antibodies were incubated for 30 min at room temperature. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac) using the Bright Vision + polymer detection system (Medac) and the following settings: Enhancer for 10 min, polymer for 20 min, DAB (415192F, Medac) for 8 min. Afterwards, nuclei were stained by hematoxylin for 3 min. See Table 2 for antibody details and dilution ratios.