Figure 6.

VEGF (a, b) and IDO (c, d) expression in the sections of axillary lymph nodes (ALNs), using IHC staining at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to VEGF (Dako, M7273) at a 1 : 50 dilution for 30 mins at RT, MAbs to IDO (Abcam, ab55305) at a concentration of 0.75 μg/ml for 15 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) Low level of expression; (b, d) high level of expression. The H score (% of positive cells (brown membrane/cytoplasmic-stained cells) × intensity of staining (1 to 3)) was used to assess the level of expression; low was ≤100 and high was >100. Scoring performed on nonmetastatic areas of a whole ALN section (7–10 HPFs).