Immunohistochemical Analysis of Immune Cells

Immunohistochemistry (IHC) was described in our published study [14 (link)]. Undergoing deparaffinized, rehydrated, and antigen retrieval (EDTA 9.0), slides were blocked with 3% hydrogen peroxide and 5% goat serum, then being incubated overnight at 4 °C with primary antibodies. In the next day, slides were incubated with enhancer and secondary antibody at room temperature. A DAB Substrate Kit was used for chromogenic reaction. Finally, the sections were stained with hematoxylin, then dehydrated, cleared, and evaluated. Primary antibodies include anti-CD4 antibody (ab133616, Abcam, USA, 1:100), anti-CD8 antibody (SP16, Thermo Fisher Scientific, USA, 1:100), anti-CD68 antibody (KP1, Thermo Fisher Scientific, USA, 1:200), and anti-CD11c antibody (EP1347Y, Abcam, USA, 1:200). Immunostaining was evaluated under light microscopy at 400× magnification by two independent pathologists. The absolute number of immune cells was counted manually. The total number of stained immune cells (in the central tumor and peritumoral stroma) was included in the analyses.