Formalin-fixed TMA sections were freshly cut (2–3 μm) and mounted on superfrost specimen slides (Thermo Fisher). Next, dewaxing was carried out with xylene and the tissue sections were gradually rehydrated. Antigen retrieval was achieved by pressure cooking in the autoclave at pH6 and under hyperfrequency wave of 360 W at 125 °C for 8 min. MAb SWA11 was diluted 1:100 and SN3b was diluted 1:50, each using a modul buffer from Medac (TA-250-PM). The immunohistochemical reaction was visualized using the detection system C-DPVB 500 HRP by Medac (all procedures were conducted according to the instructions of the manufacturer).
Positive controls, consisting of tissue samples with known positivity for the antibody, and negative controls (i.e. reactions lacking the primary antibody), were performed in parallel for each TMA slide. Expression intensity was examined in a semiquantitative manner (score 0: no staining, score 1: weak, score 2: moderate and score 3: strong staining). For statistical analyses, cases with moderate to strong expression were bundled in a ‘high expression’ and cases with negative or weak expression in a ‘low expression’ group.
Positive controls, consisting of tissue samples with known positivity for the antibody, and negative controls (i.e. reactions lacking the primary antibody), were performed in parallel for each TMA slide. Expression intensity was examined in a semiquantitative manner (score 0: no staining, score 1: weak, score 2: moderate and score 3: strong staining). For statistical analyses, cases with moderate to strong expression were bundled in a ‘high expression’ and cases with negative or weak expression in a ‘low expression’ group.