Bright vision polymer detection system

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.

A total of 4 × 10⁴ tumour cells in PBS/1.5% BSA were cytospun at 12000 g for 5′ onto glass slides and air‐dried for 15′. Signal detection was performed semiautomatically in the Autostainer 480 S (Medac, Wedel, Germany) using the Bright Vision+ polymer detection system (Medac) and the following settings: anti‐CD30 primary antibody (BER‐H2, dilution 1:200, Dako, Eching, Germany) for 20′, enhancer for 10′, polymer (Poly‐HRP‐Goat anti‐mouse/‐rabbit‐IgG) for 20′, 3,3′‐diaminobenzidine (DAB) (415192F, Medac) for 8′. Nuclei were stained by haematoxylin for 3′.