Expression of several Eph ligands in normal skin (ephrin-A3, ephrin-B1, and ephrin-B2). (A) Expression of ephrin-A3, ephrin-B1, and ephrin-B2 by day-11 CD34⁺-derived DCs cultured in the presence of GM-CSF and TNF-α. Ephrin-A3, ephrin-B1, and ephrin-B2 expression was analyzed by cytofluorometry with anti-ephrin-A3 (K19), anti-ephrin-B1 (C18), or anti-ephrin-B2 (P20) polyclonal antibodies directed against intracytoplasmic epitope, after DC permeabilization with saponin, as described in “Materials and methods.” Dotted-line overlay histograms show negative fluorescence control with an antibody of unrelated specificity, and bold dotted-line overlay histograms represent staining after addition of the blocking peptides. (B) Immunocytochemistry analysis of ephrin-A3 (ii), ephrin-B1 (iii), and ephrin-B2 (iv) on day-11 CD34⁺-derived DC cytospins. Staining was performed with anti-ephrin-A3 (K19), anti-ephrin-B1 (C18), or anti-ephrin-B2 (P20) polyclonal antibodies. The specificity of the staining was confirmed by the absence of detection in the presence of the immunization peptide (data not shown). The negative control (i) represents a polyclonal antibody of unrelated specificity. (C) Immunohistochemistry analysis of ephrin-A3 (ii), ephrin-B1 (iii), and ephrin-B2 (iv) expression in vivo. Staining was performed on normal serial skin sections with anti-ephrin-A3 (K19), anti-ephrin-B1 (C18), or anti-ephrin-B2 (P20) polyclonal antibodies. The negative control (i) represents a polyclonal antibody of unrelated specificity (original magnification × 200 for B and C).