Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections.
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.
Hosoya
Akihiro
A
Department of Oral Histology, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano, 399-0781, Japan. hosoya@po.mdu.ac.jp
Hoshi
Kazuto
K
Sahara
Noriyuki
N
Ninomiya
Tadashi
T
Akahane
Syoji
S
Kawamoto
Tadafumi
T
Ozawa
Hidehiro
H
D000818:Animals; D001857:Bone Matrix / Q000737:chemistry* / Q000187:drug effects; D024042:Collagen Type I / Q000032:analysis; D003648:Decalcification Technique; D004492:Edetic Acid / Q000494:pharmacology; D004577:Electron Probe Microanalysis; D005269:Femur / Q000737:chemistry* / Q000187:drug effects; D005557:Formaldehyde / Q000494:pharmacology; D005629:Frozen Sections; D007150:Immunohistochemistry / Q000379:methods; D058574:Integrin-Binding Sialoprotein; D008852:Microradiography; D053495:Osteopontin; D011108:Polymers / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012795:Sialoglycoproteins / Q000032:analysis; D013194:Staining and Labeling; D016707:Tissue Fixation / Q000379:methods
D024042:Collagen Type I; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D011108:Polymers; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D005557:Formaldehyde; D004492:Edetic Acid; C003043:paraform