Cd4 for t helper

IHC was performed on 4 µm serial FFPE tonsil sections to characterize cell populations with the following antibodies: CD8 for cytotoxic T lymphocyte (CTL) (clone SP57, Ventana Roche, Tucson, USA), Granzyme B (GrB) for activated cytotoxic cells (clone GB7, AbD Serotec, Oxford, UK), CD68 for macrophages (clone KP-1, Ventana Roche), IL10 for anti-inflammatory cytokine-productive cells (Abcam), Foxp3 for regulatory T lymphocytes (Treg) (Abcam, Cambridge, UK), PD1 (CD279) for T cell-negative regulator (AbD Serotec), CD56 for NK cells (Leica, Buffalo, IL, USA) and CD4 for T helper (Th) (Ventana Roche). Lymph node reactive hyperplasia tissue was used as positive control. Negative controls for each case consisted in substituting the primary antibody with antibody dilution buffer and an isotype control. The stains were developed using diaminobenzidine (DAB).
Given the fact that we previously characterized viral antigen expression in lymphocytes at germinal center (GC), interfollicular (IF) and subepithelial (SubEp) regions, the same approach was used for microenvironment characterization at those three histological regions [19, (link)20] (link).
All cell markers were observed and counted by two pathologists in serial slides on the basis of the best-preserved areas around LMP1+ and LMP1-zones. The results were expressed as immunopositive cells nº/100 total cell nº.