Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections
Effects of fixation and decalcification on the immunohistochemical localization of bone matrix proteins in fresh-frozen bone sections
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DOI：
10.1007/s00418-005-0791-4
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发表时间：
2005-06-01
影响因子：
2.3
通讯作者：
Ozawa, H
中科院分区：
文献类型：
--
作者：
Hosoya, A;Hoshi, K;Ozawa, H
为了检查晶体脱位的骨基质蛋白的稳定性，在固定和脱钙的不同阶段研究了I型胶原、骨唾液蛋白和骨桥蛋白的免疫定位。将4周龄大鼠股骨快速冷冻，并在不固定或脱钙的情况下切片。此后，以下或旁路固定在4%的多聚甲醛，这些部分脱钙在5%EDTA 0-5分钟。脱钙前，显着的骨基质的不透射线性观察接触显微放射照相术（CMR）的图像，和电子探针显微分析（EPMA）表现出强烈的本地化磷和钙。在未脱钙的固定和未固定切片中，骨基质蛋白的免疫定位几乎仅限于类骨质。脱钙1分钟后，CMR图像中的不透射线性明显降低，EPMA观察到的磷和钙减少，5分钟脱钙后完全消失。脱钙3-5分钟后，未固定切片显示这些蛋白质在骨基质中免疫定位，但在类骨质中检测不到。然而，固定切片表明，这些都发现在骨基质和类骨质。目前的研究结果表明，骨基质蛋白包埋在钙化基质中，钙化基质与水环境分离，并且它们几乎不移动，可能是由于彼此牢固结合。相反，类骨质中的基质蛋白在脱钙后会丢失，因为它们可能与分散的磷灰石晶体结合，而不是彼此结合。
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.