Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration
Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration
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DOI：
10.1186/s12861-016-0102-4
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发表时间：
2016-01-19
影响因子：
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通讯作者：
Witten, P. E.
中科院分区：
文献类型：
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作者：
Bensimon-Brito, A.;Cardeira, J.;Witten, P. E.
背景：矿化的正确评估是骨骼发育、维护和再生研究的基础。目前观察斑马鱼矿化组织的方法依赖于：1）固定标本； 2) 放射线照相和 mu CT 技术，最终分辨率受到限制；或 3) 使用荧光染料进行活体染色，与绿色荧光蛋白 (GFP) 标记细胞的信号无法区分。茜素化合物，无论是茜素红 S (ARS) 还是茜素络合酮 (ALC)，长期以来一直被用来对所有脊椎动物群体的固定标本中的矿化骨骼进行染色。最近的工作在斑马鱼和青鳉中使用了 ARS 活体染色，但并非基于一致的方案。通过在水中添加 ARS 实现的 ARS 活体染色是否会影响幼年和成年斑马鱼的骨形成，这是一个基本问题，因为 ARS 已被证明可以抑制哺乳动物的骨骼生长和矿化。结果：在这里，我们提出了一种对斑马鱼矿化结构进行活体染色的方案，低 ARS 浓度不会影响骨矿化，即使在重复的 ARS 染色事件之后，这一点通过在荧光灯下仔细成像得到证实。骨骼发育的早期和晚期阶段同样不受这种活体染色方案的影响。从所有测试浓度来看，0.01% ARS 能够正确检测骨钙沉积物，而不会给鱼类带来额外的应激。结论：所提出的 ARS 活体染色方案可以与骨骼组织相关的 GFP 荧光相结合，因此代表了一种用于体内矿化结构监测的强大工具。我们提供野生型和表达 GFP 的转基因斑马鱼的例子，用于内骨骼发育和皮鳍条再生。
Background: The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and mu CT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals.Results: Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish.Conclusions: The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.