Immunostaining of pDCs in human tonsil. Top row, left: Clusters of pDCs (arrowed and at higher magnification in the inset) lying close to vessels outside a lymphoid follicle (Foll.) in the T-cell–rich region are strongly stained for the adaptor protein CD2AP (immunoperoxidase technique, hematoxylin counterstain). Right: Double immunoenzymatic labeling for CD2AP (brown) and the B-cell–associated transcription factor BCL11A (blue) confirms that both molecules are present in the same cells (no counterstain). Middle row: Coexpression of 3 B-cell–associated transcription factors, namely ICSBP (brown), E47 (red), and FOXP1 (brown), in pDCs expressing CD2AP (blue or brown). Note that FOXP1 is also expressed in B cells and in endothelial cells in a vessel (Vess; double immunoenzymatic staining, hematoxylin counterstain for E47 + CD2AP staining). Third row: The B-cell–associated transcription factors BCL6 (brown) and PAX5 (blue) are expressed in B-cell follicles (Foll.), but are absent from extrafollicular pDCs (identified by immunostaining for the cytoplasmic marker CD2AP in blue or brown). Scattered BCL6- and PAX5-positive B-cell nuclei among the pDCs are arrowed (no counterstain). (Images were acquired on a Nikon Eclipse E800 microscope [Nikon, Tokyo, Japan] equipped with 10×/0.45 Plan Apo or 20×/0.7, 40×/0.95, and 60×/1.4 Plan Fluor objective lenses [Zeiss], using a Zeiss Axiocam digital camera [Zeiss, Oberkochen, Germany], Axiovision 3 image acquisition software [Zeiss], and Adobe Photoshop 7 image processing and manipulation software [Adobe, San Jose, CA]).