FIG. 4.

Detection of hCAP-18 on the surface of spermatozoa. (A) Flow cytometry showing the presence of hCAP-18 on the surface of spermatozoa. (B) Flow cytometry of spermatozoa, where the specific primary antibody has been replaced by nonimmune IgG at the same concentration, resulting in loss of labeling. (C) Immunocytochemistry showing the presence of hCAP-18 on the surface of spermatozoa. Binding of specific antibody is visualized by a peroxidase reaction, resulting in brown deposits. Magnification, ×25. (D) Higher magnification (×40 objective) of spermatozoa immunolabeled for hCAP-18, showing immunoreactivity with a predominance in the neck and tail region of the spermatozoa. (E) Control, where the primary antibody has been replaced by nonimmune IgG, resulting in loss of labeling. A ×25 objective was used. The cells were counterstained with Mayer's hematoxylin. (F) Western blot of lysates of washed spermatozoa. An 18-kDa immunoreactive double band corresponding to hCAP-18 in seminal plasma (1) as well as in homogenates obtained from washed spermatozoa (2) is seen. In addition, the lane holding lysed spermatozoa shows a faint 14-kDa band and a stronger 5-kDa band corresponding in size to the cathelin fragment and LL-37, respectively (arrows).