caa a22 4500 469030984 CHVBK 20180323132743.0 cr unu---uuuuu 170328e19920201xx s 000 0 eng 10.1007/BF01082443 doi (NATIONALLICENCE)springer-10.1007/BF01082443 Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization [Elektronische Daten] [Minoru Takagi, Masao Maeno, Yasuki Takahashi, Kichibee Otsuka] Summary: The present study utilized biochemical and immuno-and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific forN-acetyl-d-glucosamine,N-acetylneuraminic acid, andN-acetyl-d-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl. For comparison with the second extraction step, ethanolic trimethylammonium EDTA (ethanolic EDTA) was substituted for aqueous EDTA. Based on protein staining and Western blot analysis of SDS-polyacrylamide gel electrophoresis of each extract using 9-A-2 and WGA, retention of mineral-binding proteins extractable from fresh specimens with aqueous EDTA was greatly increased in tissue when ethanolic EDTA was used. Their retention was even greater with prior aldehyde fixation. Maximum retention with no detectable solubility of 9-A-2 and WGA reactive proteins was obtained after ethanolic EDTA extraction of aldehyde-fixed specimens, which concomitantly provided the strongest immuno- and lectin staining. These results indicate that this combined method dramatically improves retention of PGs and glycoproteins during demineralization of bone tissues and provides the best method for localizing these glycoconjugates. Chapman & Hall, 1992 Takagi Minoru Department of Anatomy, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, 101, Tokyo, Japan aut Maeno Masao Department of Biochemistry, Nihon University School of Dentistry, 101, Tokyo, Japan aut Takahashi Yasuki Department of Anatomy, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, 101, Tokyo, Japan aut Otsuka Kichibee Department of Biochemistry, Nihon University School of Dentistry, 101, Tokyo, Japan aut The Histochemical Journal Kluwer Academic Publishers 24/2(1992-02-01), 78-85 0018-2214 24:2<78 1992 24 10735 https://doi.org/10.1007/BF01082443 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF01082443 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Takagi Minoru Department of Anatomy, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, 101, Tokyo, Japan aut NATIONALLICENCE 700 1- Maeno Masao Department of Biochemistry, Nihon University School of Dentistry, 101, Tokyo, Japan aut NATIONALLICENCE 700 1- Takahashi Yasuki Department of Anatomy, Nihon University School of Dentistry, 1-8-13, Kanda-Surugadai, Chiyoda-ku, 101, Tokyo, Japan aut NATIONALLICENCE 700 1- Otsuka Kichibee Department of Biochemistry, Nihon University School of Dentistry, 101, Tokyo, Japan aut NATIONALLICENCE 773 0- The Histochemical Journal Kluwer Academic Publishers 24/2(1992-02-01), 78-85 0018-2214 24:2<78 1992 24 10735 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer