Immunohistochemical Analysis of Immune Markers

Standard indirect immunoperoxidase procedures were used for immunohistochemistry (IHC; ABC-Elite, Vector Laboratories, Burlingame, CA). Slides were dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum (DakoCytomation, Carpinteria, CA) for 20 min and incubated with primary antibody at room temperature. Primary antibodies used were specific for OX40 (polyclonal anti-CD134/OX40, ab119904, Abcam, Cambridge, UK), CD8 (clone C8/144B, DakoCytomation, Switzerland) and FOXP3 (clone 236A/E7, Abcam, Cambridge, UK) [7 (link), 10 (link)]. Subsequently, sections were incubated with peroxidase-labelled secondary antibody (DakoCytomation) for 30 min at room temperature. To visualize the antigen, sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen (DakoCytomation) for 30 min, and counterstained with Gill's hematoxylin.