Quantifying Immune Cell Types in Skin Tissue

Tissue sections of the paraffin-embedded biopsies were stained with haematoxylin–eosin (HE) for the detection of eosinophils, toluidine blue staining for mast cells and two immunohistochemical stainings (CD3 staining for T-cells and CD20 staining for B-cells), based on Dreesen et al. [31 (link)]. In short, skin tissue sections of 4 μm were mounted on APES-coated slides, blocked with H₂O₂ and stained with polyclonal rabbit anti-human CD3 (Dako, Belgium) or rabbit anti-human CD20 (Sigma-Aldrich, USA) antibodies. T- and B-cells were visualized by adding peroxidase labelled goat anti-rabbit antibodies (Dako, Belgium), diaminobenzidine tetrahydrochloride (DAB; Dako, Belgium) and by performing a counterstaining with haematoxylin. Eosinophils, mast cells, T-cells and B-cells were quantified by taking two random pictures per tissue slide at 400× magnification on a LEICA light microscope and counting the positive cells on two tissue slides per animal. Results were expressed as the number of cells per 10⁵ μm² tissue surface.