Matskási István (szerk.): A Magyar Természettudományi Múzeum évkönyve 87. (Budapest 1995)

Józsa, L. ; Pap, I.: Histochemical and immunohistochemical analysis of mummy skin

antibody reacts not only to a specific antigen but to other, chemically similar proteins, too. Antigens can be detected with great certainty by immunohistochemical methods thanks to the extraordinary specificity of these methods. Immunohistochemical investigations were started with blood group characteristics of ancient material. First ABO, and later Rh, MN and P blood groups became detectable. The two later ones were identified only in mummy tissues, but the others in skeletalized material, too (LENGYEL 1970, CRAINIC et al. 1989). The number and variety of detectable substances and pathogens is constantly growing. More than one thousand various antigens (proteins, cellular markers, receptors, etc.) are recognisable on recent material. Not a fraction of these was described for mummy tissue and immunohistochemistry was only scarcely if ever utilized for paleohistology. Our purpose was to determine the tissue components and cell markers that can be detected in mummy tissues. MATERIAL AND METHODS We analysed the scalps of two Egyptian mummies from the 1st century A. D. housed in the Department of Anthropology of the Hungarian Natural History Museum, Budapest. Their detailed paleopathological examination was carried out by MÉREI & NEMESKÉRI (1958). Samples of approximately 10x6 mm were taken from the preauricular regions (regio parotidea) of the mummies' scalps. The skin pieces were fixed in Ruffer solution (formalin, water, sodium carbonate). They were softened and rehydrated for three days, then they were washed in tap water, and they were subjected to the routine histological paraffin embedding utilizing a Sandon-Elliot automatic processor. The material was oriented and cut in a way that the full thickness of epithelium and corium layers was contained within the specimen. Histological methods: Haematoxilin-eosin (HE), picrosyrius, Gömöri's reticulin staining, Mallory-trichrom, Masson-trichrom staining were carried out for histological evaluation. Melanin pigment was indicated by the method of Masson-Fontana. Myelinated neurofibrils were indicated by Luxol-Fasl blue staining. Elastic fibres were stained by orcein and resorcin fuchsin. All sections were analysed and photographed by light and polar microscopes. Histochemical methods: Proteins with aldohexose contents (glycoproteins, proteoglycans) were indicated by periodic Schiff reaction (PAS), while glycosaminoglycans by alcian blue (pH 0.5 pH 2.5) and alcian blue PAS reactions by toluidin blue staining. Prussian blue reaction was used in an attempt to indicate siderous materials. Immunohistochemical methods: Sections were digested in 37 °C, 0.1% pepsin solution (Merck, Darmstadt) for two hours. Recent technology dictates a hydrogen peroxide solution to inhibit tissue peroxidase as a next step. As we supposed that there were no active enzymes in mummy tissues, first we passed over this step. We were astonished to find a large proportion of red blood cells with various strengths of peroxidase activity. This accident led us to detect enzymes in mummy tissue for the first time. Observing this fact we stopped to pass over hydrogen peroxide treatment before immunohistochemical reactions in all other analysis processes. Antigen-antibody reaction took 18 hours in a wet chamber at room temperature. The location of the reaction was marked by ovidin-biotin-peroxidase (Vector Laboratories Burlingames, USA) reaction. The antibodies and dilutions applied were summarized in Table 1. Table 1. Immunohistochemical reagents and solutions applied Antibody Manufacturer Solution Fibronectin Dako/Copenhagen 1 500 Laminin Sigma/ST Louis 1 500 Collagen Type I Biogenesis/Bourhemouth 1 1500 Collagen Type III Biogenesis/Bourhe mouth 1 500 Collagen Type IV Biogenesis/Bourhemouth 1 200 Tenascin Telios/San Diego 1 300 Thrombospondin Oncogene/Manasset 1 1000 Vitronectin Chemicon/Temecula 1 250 EMA (E 29) Dako/Copenhagen 1 200 Cytokeratin (LP 34) Dako/Copenhagen 1 250 Vimentin (V.9) Dako/Copenhagen 1 200 Desmin (D33) Dako/Copenhagen 1 200