Fig. 2

CD4⁺ (a, b), CD8⁺ (C, D) T lymphocytes, FOXP3⁺ Tregs (e, f) and CD56⁺ NK cells (G, H) in the sections of metastatic tumours, 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 CD4 (Dako, M7310) at a 1:80 dilution for 30 mins at RT, 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, g low level of CD4⁺, CD8⁺ T cell, FOXP3⁺ Treg, CD56⁺ NK cell infiltration respectively; b, d, f, h: high level of CD4⁺, CD8⁺ T cell, FOXP3⁺ Treg, CD56⁺ NK infiltration respectively. The average number of brown membrane-stained cells (CD4⁺, CD8⁺ T cells, CD56⁺ NK cells) and brown nuclear-stained cells (FOXP3⁺ Tregs) regardless of intensity, in contact with tumour cells or within tumour cell nests per HPF was counted. MTu: Metastatic tumour nest; LN: Lymphoid tissue