Immunohistochemical localization of calmodulin in normal human epidermis

Abstract

Calmodulin, an intracellular Ca 2 +-binding protein, was first found to be a Ca 2 +-dependent activator protein of cyclic nucleotide phosphodiesterase [2], and a number of Ca 2+-dependent processes are regulated by calmodulin in vitro [3]. In the skin, although the epidermal cells actually contain a considerable amount of calmodulin [1, 4, 6-8], its role in the epidermis is not clearly known. With regard to the distribution of calmodulin in normal epidermis, there are few reports [5, 9] and these indicate quite different distributions of calmodulin. Where does calmodulin exist in normal epidermis? We studied the distribution of calmodulin in normal human epidermis both in paraffin-embedded and frozen sections, using the avidin-biotin glucose oxidase complex method. Samples of normal human skin were obtained from healthy volunteers after local anaesthesia. The specimens were divided, and processed for routine formalin fixation or were frozen in OCT compound. We used the ABC kits with glucose oxidase as the enzyme marker (Vector Laboratories Inc., Burlingame, Calif.). After deparaffinization, paraffin sections were incubated with 100% methanol containing 0.3% H20 2 for 30 rain. Each section was rinsed with 50 mM Tris HC1 containing 0.15% NaC1 (pH 7.6) (TBS) and incubated with normal rabbit serum for 20 rain. After washing with TBS, the sections were incubated for 30 rain with sheep anti-bovine testis calmodulin IgG diluted 50-fold (Biomedical Technologies Inc., Stoughton, Mass., USA). The sections rinsed with TBS were then incubated with biotinylated anti-sheep IgG for 30 rain. After incubation for 30 rain with avidin DHbiotinylated glucose oxidase H complex, the sections were reacted with iodonitrotetrazolium or tetranitroblue tetrazolium as substrates. For staining the frozen sections, the procedure employed was the same as above except for the deparaffinization step.