Keratinocytes and Langerhans cells from skin expression isolated ex vivo. (A) Immunohistochemistry analysis of EphA2 expression in vivo. Double staining was performed on normal serial skin sections with anti-EphA2 polyclonal antibody and the anti-DCGM4 mAb, specific for Langerin. 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 represents a rabbit polyclonal antibody and an mAb of unrelated specificity. Original magnifications, × 200 (left and middle panels) and × 400 (right panel). (B) Expression of EphA2 by keratinocytes from normal skin isolated ex vivo. Flow cytometry staining was performed with anti-EphA2 polyclonal antibody or with anti-EphA2 polyclonal antibody plus the peptide used for immunization, after DC permeabilization with saponin, as described in “Materials and methods.” The negative control represents a rabbit polyclonal antibody of unrelated specificity. The bar corresponds to the region of the positive EphA2+ cells. Results are representative of 3 experiments. (C) Expression of EphA2 by Langerhans cells from skin isolated ex vivo. Langerin and EphA2 expression was analyzed by flow cytometry on enriched Langerhans cells with an anti-EphA2 polyclonal antibody or with mAb DCGM4, specific for Langerin (open histogram), and compared with species-specific unrelated antibodies (filled histogram). Results are representative of 3 experiments. (D) Double staining of another Langerhans cells preparation with surface FITC-CD1a and intracellular anti-EphA2 polyclonal antibody revealed with PE-antirabbit IgG.