Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections |
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Authors: | Email author" target="_blank">Akihiro?HosoyaEmail author Kazuto?Hoshi Noriyuki?Sahara Tadashi?Ninomiya Syoji?Akahane Tadafumi?Kawamoto Hidehiro?Ozawa |
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Institution: | (1) Department of Oral Histology, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan;(2) Department of Menicon Cartilage and Bone Regeneration, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;(3) Institute for Dental Science, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan;(4) RI Research Institute, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan |
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Abstract: | To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization of type I collagen, bone sialoprotein, and osteopontin was investigated during different stages of fixation and decalcification. Four-week-old rat femurs were rapidly frozen, and were sectioned without fixation or decalcification. Thereafter, following or bypassing fixation in 4% paraformaldehyde, these sections were decalcified in 5% EDTA for 0-5 min. Before decalcification, marked radiopacity of bone matrix was observed in contact microradiography (CMR) images, and electron probe microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In fixed and unfixed sections without decalcification, immunolocalization of bone matrix proteins were almost restricted to osteoid. After 1 min of decalcification, reduced radiopacity was apparent in the CMR images, and less phosphorus and calcium was observed by EPMA, which completely disappeared by 5 min decalcification. After 3-5 min of decalcification, unfixed sections showed that these proteins were immunolocalized in bone matrix, but were not detectable in osteoid. However, fixed sections demonstrated that these were found in both bone matrix and osteoid. The present findings suggest that bone matrix proteins are embedded in calcified matrix which is separated from the aqueous environment and that they hardly move, probably due to firm bonding with each other. In contrast, matrix proteins in osteoid are subject to loss after decalcification because they may be bound to scattered apatite crystals, not to each other. |
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Keywords: | Fresh-frozen section Non-decalcified section Bone matrix proteins Immunohistochemistry Bone |
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