Fig. 3

CD8⁺ T cells (a, b), FOXP3⁺ Tregs (c,dD) and CD56⁺ NK cells (E, F) 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 CD8 (Dako, M7103) at a 1:100 dilution for 30 mins at RT, MAbs to FOXP3 (Abcam, ab20034) at a concentration of 20 μg/ml for 30 mins at RT, MAbs to CD56 (Dako, M7304) at a 1:50 dilution 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: low percentage of CD8⁺ T cells, FOXP3⁺ Tregs and low number of CD56⁺ NK cells respectively; B, d, d: high percentage of CD8⁺ T cells, FOXP3⁺ Tregs and high number of CD56⁺ NK cells respectively. The positively brown membrane-stained cells (CD8⁺ T cells) and brown nuclear-stained cells (FOXP3⁺ Tregs) in non-metastatic paracortical areas of ALNs were quantified as the average % of all cells (5 HPFs). CD56⁺ NK cells were quantified as average number of cell count per HPF in non-metastatic para-cortical areas of ALNs with the greatest accumulation of the positively brown membrane-stained cells