Figure 5

Immunohistochemical detection of CD20⁺ lymphocytes with intracellular staining for IL-17. (A) Peripheral blood mononuclear cells were isolated from peripheral blood and stimulated with CytoStim followed by Brefeldin A and stained histochemically with Diff-Quick™. (B-I) Cells were then phenotyped for IgG1-PE isotype control (C), CD20-PE (E), or CD19-PE (H). The cells were then fixed and permeabilized and stained for IL-17-FITC, as seen in (C), (F), and (I), respectively. The cells were mounted on slides with antifade fluid supplemented with DAPI for nuclear staining (B, D, and G). Arrows in the middle column of panels depict triple-stained CD20⁺ (red) lymphocytes, which are IL-17⁺ (green). The right-hand column of panels depicts CD19⁺ lymphocytes (red), which are IL-17^-. Images were viewed at ×1,000 with an Olympus BX60 microscope with C-mount and a Nikon S10 digital camera. The size bar represents 10 μm unless otherwise stated. All specimens were processed at the same time, as described in Materials and methods. At least 10 fields of view were examined for each panel. DAPI, 4',6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; IL, interleukin; PE, phycoerythrin.