Immunostaining of pDCs in blood and bone marrow. Upper panel: Immunoperoxidase staining of normal peripheral blood mononuclear cells (cytospin preparation) shows rare cells expressing the pDC marker CD2AP. Double labeling (right) shows that these cells do not express CD3 or CD20 (double immunofluorescence), but they coexpress (as in tissue) CD2AP (brown) and BCL11A (red) (double staining). Immunostaining of cytospin preparations of pDCs isolated from peripheral blood using anti–BDCA-4 shows that most cells express CD2AP and also the pDCs marker CD123 (and no staining is seen in a negative control). CD2AP is also expressed by pDCs isolated from peripheral blood using anti–BDCA-4 (cytospin preparation; DAPI counterstain for the immunofluorescence preparations, hematoxylin counterstain for the immunoenzymatic preparations). Lower panel: Scattered pDCs (arrowed) are seen in a bone marrow trephine (top row) immunostained for CD2AP and glycophorin (in brown and red, respectively). In the lower row, examples are shown at high magnification of bone marrow pDCs coexpressing CD2AP (brown) and BCL11A (red; hematoxylin counterstain). (Images were acquired on a Nikon Eclipse E800 microscope [Nikon, Tokyo, Japan] equipped with 60×/1.4 and 100×/1.3 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].)