Using a serum raised in rabbits against a calcium binding protein extracted from rat skin, the cutaneous localization of this protein was studied by indirect immunofluorescence. The skin calcium binding immunoreactivity was found in the epidermis but not in the dermis; it was localized in the cytoplasm of the basal cell layer of both skin and malpighian mucosa. Thel'e was not species specificity; this allowed the tracing of the protein in human epidermis as well where it was also expressed only

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... in the basal cells. This is the first demonstration of the unique localization of a specific protein within the cytoplasm of the basal cell layer of the epidermis. This localization may help to elucidate the physiological role of this protein. ' Calcium binding protein (CaBP) is one of the known molecular e xpressions of the hormonal action of the cholecalciferol (vitamin D) on intestine. In this organ, CaBP is found in the cytoplas m of absorptive cells in mammals a nd birds [1, 2]. The rol e of intestinal CaBP is supposed to be either in calcium transport [3] or in " buffering" intracellular calcium [4]. Recentl y we (5] h a ve de monstrated t hat a vitamin D de pe nde nt CaBP was prese nt in r at skin. Molecular weight a nd chromatographic properties of this cutaneous CaBP (SCaBP) we re similar to intestinal CaBP (iCaBP). However , it was immunologically different. Since SCaBP was isolated from t iss ue specimens containing both dermis a nd epidermis, th e distribution within the s kin of this protein was not known. Knowing the localization of SCaBP in the skin may h elp elucidate its fun ction [3). In this st udy immunohistological tracing of SCaBP in the skin of several species was p erform ed. Since SCaBP h as b een found to vitamin D de pendent [5] and since DVB is r esponsible for the photobiosynt hesis of vitamin D3 in t h e epidermis [6] we also s tudied unexposed m alpighian (mu cosal) epithelial for the content a nd distribution of SCaBP. MATERIALS AND METHODS Antisera A rabbit serum co ntaining antibodies against rat SCaBP. was obtained as previously desc ribed [5] . Briefly, SCaBP from whole Sprangley albinos rats skin was extracted by homogenization and purified by chromatography. An antiserum to the pW'ified protein was raised by Manuscrip t T h is work was supported by funds from Co nseil Scien tifiqu e Faculte Necker-Enfants-Malades INSERM ATP 67 78 99, CNRS AI 033 395 and DGRST 77 70668. Reprint req uests to: J. H. Saurat Secteur de Dermatologie experimentale Lab. Path, Exp. UER Nec ker-Enfants-Malades 156, ru e de Vaugirard 75730 Paris Cedex 1.5 France. Abbreviations: iCABP: intestinal calcium binding protein SCaBP: skin calcium binding protein. immunization of white rabbits. Its specificity and cross reactivity were tested with an Ouchterlony's dou ble immunodiffusion tec hniqu e; briefly a single precipita tion line was obta ined with both crude skin extract and purified SCaBP [5] . Preimmune serum a nd a rabbit antiserum to in testinal CaBP (iCaBP) [3] were used as controls. A bsorption Rabbit antiserum to rat SCaBP was absorbed wit h the purified protein [5] ; SCaBP (at a concentra tion of 2 and 20 mg/ ml owing to the strength of the antiserum) was incubated with the antiserum dilu ted 1/ 10 to 1/640 for 1 hI' at + 37°C and 24 hI' at + 4°C then centrifugated at 2700 g for 1 hI' and the supernantant was taken as reagent for IIF procedure. In a same way, control absorptions were made with rabbit brain and liver extracts (20 mg/ ml) Ca ppel Laboratories. USA). I ndirect Immunoflu. orescen ce (IIF) Back skin and oesophagus specimens were obtained from Sprangley alb ino rats, New Zealand rabb its, Hartley guin ea-pigs and C3H inbred mice. Sagittal section from the inferior lip of the same animals was obtained in order to study the parakeratotic and mu cosal sides of these substrates as previously described [7] . Normal human skin specimens were provided by the plastic surgery unit. One-and lO-day-old neonatal mouse skin, ad ul t rat, and guin ea pig in tesinal specimens were included in the study. The specimens were snap fi'ozen in liquid ni trogen and kept at -30°C within a week, they were sectioned with a cryostat microtome set at 4 fLM.