Immunostaining of human tonsil for signaling molecules in pDCs. Top row: BLNK is clearly present in a cluster of pDCs (circled and at higher magnification in the inset) lying close to a lymphoid follicle (Foll.) (immunoperoxidase staining, hematoxylin counterstain). Double immunofluorescent labeling (with CD2AP) shows that pDCs also express the signaling molecules Syk and Btk. (DAPI [4′6-diamidine-2-phenylindole 2 HCl] counterstain for Syk and CD2AP). Middle row: pDCs, identified by double staining for the transcription factor BCL11A (blue), express the transmembrane adaptor protein LIME (brown) but are negative for 2 other T-cell–associated signaling molecules (TRIM and SLP76—both brown). The arrows indicate LIME-positive BCL11A-negative T cells (no counterstain). Third row, left: A cluster of pDCs (circled and at higher magnification, center) lying adjacent to a lymphoid follicle (Foll.) coexpress DAP12 (brown) and the transcription factor BCL11A (blue). Macrophages in the follicle (arrowed) express DAP12 alone (no counterstain). Right: TCB1B4 (a GTPase-activating protein) is also strongly expressed by clusters of pDCs (circled in low power view). Note expression of TCB1B4 in a B-cell follicle (Foll.) (immunoperoxidase staining, hematoxylin counterstain). (Images were acquired on a Nikon Eclipse E800 microscope [Nikon, Tokyo, Japan] equipped with 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]).