Generation of a polyclonal antibody to mouse endosialin. Antibody 171 specifically recognizes mouse endosialin in cell extracts (A) and in tissue sections (B, C). (A) Immunoblot analysis of cell extracts prepared from mEs-transfected 293T cells shows three bands, corresponding to the known 90-kD, 110-kDa and 160-kDa protein species. These bands are not seen in extracts prepared from non-transfected 293T cells, and they are abolished by preincubation with the immunizing peptide 171 (293T mEs + peptide). The positions of molecular size markers are indicated on the right. (B) Immunocytochemistry of mEs-transfected 293T cells (293T mEs) shows cell membrane staining in a subset of cells, which is not seen with a negative control IgG or following incubation with Ab 171 pre-incubated with the immunizing peptide. Cells were stained by the ABC immunoperoxidase method with hematoxylin counterstaining. (C) Immunohistochemical staining of paraffin-embedded sections from an E12.5 mouse embryo. Using Ab 171, endosialin is detected in endothelial cells of a blood vessel (arrow) and in scattered mesenchymal cells in the perineural space; no staining is seen with a negative control IgG or with Ab 171 pre-incubated with the immunizing peptide. Staining of selected cardiovascular structures in a E10.5 mouse embryo with Ab 171 to endosialin (D, E) and pan-endothelial marker CD31 (F, G) revealed strong expression of endosialin and CD31 along the endothelial lining of the dorsal aorta (arrows in D and F). The endothelial cells of the endocardium are endosialin-negative (E) in contrast to the strong staining seen with the anti-CD31 antibody (G, arrows). The ABC staining method was used, with hematoxylin counterstaining. Scale bars: 25 µm.