(A) Western blot analysis confirming the successful generation of Sp17 mouse monoclonal antibodies, showing binding of the antibodies to a recombinant Sp17 protein and not to a control recombinant protein. Both recombinant proteins were Escherichia coli–derived and contained a C-terminal 6-His tag. (i) Coomassie blue staining of a 10% sodium dodecyl sulfate–polyacrylamide gel. (ii) Western blot analysis demonstrating the binding of Sp17 mouse monoclonal antibodies to only recombinant Sp17 protein (M = molecular weight marker; lane 1 = recombinant Sp17 protein; lane 2 = control recombinant protein). (B) Sp17 protein was detected on normal spermatozoa (i and iii) but not on any of the peripheral blood leukocytes (ii and iv) by either immunofluorescence cytology (i and ii) or immunocytochemistry (iii and iv). (C) Immunohistochemistry showing the expression of Sp17 protein in normal testis (i). In contrast, Sp17 protein was not detected in either normal tonsils (ii) or Peyer patches (iii).