Fig. 4

IL-2 (a, b), IL-10 (c, d), IL-17 (e, f) and IFN-γ (g, h) expression in the sections of axillary lymph nodes (ALNs), using IHC staining, at 400× magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer pH 6 (20 mins). The sections were then incubated with MAbs to IL-2 (Abcam, ab92381) at a 1:500 dilution for 30 mins at RT, polyclonal Abs to IL-10 (Abcam, ab34843) at a 1:400 dilution for 30 mins at RT, polyclonal Abs to IL-17 (Abcam, ab9565) at a 1:100 dilution for 30 mins at RT, polyclonal Abs to IFN-γ (Abcam, ab9657) at a concentration of 4 μg/ml for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. a, c, e, g: low level of expression; b, d, f, h: high level of expression. The H score [% of positive cells (brown membrane/cytoplasmic-stained cells) x intensity of staining (1 to 3)] was used to assess the level of expression; low was ≤100 and high was > 100. Scoring performed on non-metastatic areas of a whole ALN section (7-10 HPFs)