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Epithelial/ectodermal plug formation in the developing nose, ear, and eye regions is followed by canalization/recanalization mediated by cell death. However, the mechanism is not well understood. Recently, substance P (SP)-mediated cell death, rather than cell apoptosis, has been reported in neuronal and non-neuronal cells. Horizontal paraffin sections of 5 human fetuses at 15-16 weeks of gestation were used to examine the entire area of the nose, ear, eye and perineum with immunohistochemistry for SP and its receptor neurokinin-1 (NK-1), and protein gene product (PGP) 9.5 and S100 protein to identify whether the positive cells had neural origins. The deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method was also conducted to identify apoptosis. Four SP antibodies were commercially obtained and compared the results. In addition, using the same antibodies for SP, those results were compared with fetal mouse heads (E14-17). Substance P immunoreactivity of one of the 4 antibodies (sc9758) was clearly found in the nasal plug, the epithelium of the anterior nasal cavity, the entire excretory tear duct, the marginal palpebral conjunctiva, the auditory meatal plug, the parotid duct, the external urethral orifice and, the preputial lamella along the future prepuce. Immunoreactivity was usually seen in enlarged round cells in humans. In fetal mouse heads, in spite of negative reaction in all these sites, the midline epithelial seam at the palate fusion and the oral epithelium especially at and near the tooth germ specifically reacted with the sc9758. Nevertheless, the other 3 antibodies did not react at any of those sites both in human and mouse fetuses. NK-1 receptor-positive cells were seen in the nose and meatal plugs and preputial lamella, but not in the tear duct. S100 protein, PGP 9.5, and TUNEL method all demonstrated negative reactivity at any sc9758-positive sites. Consequently, the present immunoreactivity of the sc9758 antibody seemed to be false positive, but it was likely to react with a specific substance in the epithelial or ectodermal cell because of the clearly restricted staining. Which substance it crossed remains unknown.

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False positive reactivity of a substance P-antibody in the ectodermal/epithelial plug of the nose, ear, eye and perineum of the human and mouse fetuses