The visible skeleton 2.0: phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on X-ray microCT
The visible skeleton 2.0: phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on X-ray microCT
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DOI：
10.1242/dev.187633
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发表时间：
2020-06-01
期刊：
影响因子：
4.6
通讯作者：
Handschuh, Stephan
中科院分区：
文献类型：
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作者：
Gabner, Simone;Boeck, Peter;Handschuh, Stephan
几十年来，用阿尔新蓝和茜素红进行透明和染色一直是脊椎动物骨骼发育成像的黄金标准。在这里，我们提出了一种基于 X 射线 microCT 成像来可视化骨骼和软骨的替代方法，该方法可以以微米分辨率收集整个发育骨骼的真实 3D 数据。我们的新方案基于乙醇固定和钌红染色，并有效对比软骨基质，如整个 E16.5 小鼠胎儿和 E14 鸡胚胎四肢所示。染色过程中骨矿物质得到很好的保存，因此整个胚胎骨骼可以高对比度成像。钌和钙的 X 射线衰减差异使得双能 microCT (microDECT) 能够对软骨基质和骨进行光谱分离。不需要清除样本。该协议简单且可重复。我们证明 E16.5 小鼠胎儿的软骨对比度足以进行快速视觉表型分析。形态骨骼参数很容易提取。我们认为所提出的工作流程是对目前可用于脊椎动物骨骼发育的定性和定量表型分析的工具包的强大且多功能的扩展。
For decades, clearing and staining with Alcian Blue and Alizarin Red has been the gold standard to image vertebrate skeletal development. Here, we present an alternate approach to visualise bone and cartilage based on X-ray microCT imaging, which allows the collection of genuine 3D data of the entire developing skeleton at micron resolution. Our novel protocol is based on ethanol fixation and staining with Ruthenium Red, and efficiently contrasts cartilage matrix, as demonstrated in whole E16.5 mouse foetuses and limbs of E14 chicken embryos. Bone mineral is well preserved during staining, thus the entire embryonic skeleton can be imaged at high contrast. Differences in X-ray attenuation of ruthenium and calcium enable the spectral separation of cartilage matrix and bone by dual energy microCT (microDECT). Clearing of specimens is not required. The protocol is simple and reproducible. We demonstrate that cartilage contrast in E16.5 mouse foetuses is adequate for fast visual phenotyping. Morphometric skeletal parameters are easily extracted. We consider the presented workflow to be a powerful and versatile extension to the toolkit currently available for qualitative and quantitative phenotyping of vertebrate skeletal development.