Immunohistochemical Analysis of Tumor Markers

Immunohistochemical localization was carried out as previously described by Scuderi et al. [6 (link)]. Tumor sections were incubated overnight at room temperature with the following primary antibodies: anti-Ki-67 (sc-23900, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), anti-IL-1β (sc-32294, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD4 (sc-13573, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD8 (sc-1177, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-CD30 (sc-19984, 1:100; Santa Cruz Biotechnology, Dallas, TX, USA). After this incubation, the sections were washed with PBS and incubated with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The reaction was revealed by a chromogenic substrate (brown DAB), and counterstaining with Nuclear Fast Red. A negative control was performed using no primary antibody; specifically, tissue was incubated with the antibody diluent alone, followed by incubation with secondary antibodies and detection reagents. For immunohistochemistry, 20× (50 µm scale bar) and 40× (20 µm scale bar) magnification are shown.