Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue
Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue
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DOI：
10.1038/nprot.2015.125
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发表时间：
2015-12-01
期刊：
影响因子：
14.8
通讯作者：
Adams，Ralf H.
中科院分区：
文献类型：
--
作者：
Kusumbe，Anjali P.;Ramasamy，Saravana K.;Adams，Ralf H.
高分辨率共聚焦成像是分析原位细胞三维结构和详细空间分布的重要工具。然而，骨骼组织的成像由于其钙化的性质而在技术上仍然具有挑战性。在这里，我们描述了一个协议，允许高分辨率成像的骨骼组织与保存细胞形态和组织结构。该程序涉及组织固定、脱钙和小鼠骨骼组织的冷冻切片以生成厚切片。通过该程序生成的厚切片不仅与遗传表达的荧光蛋白的分析兼容，而且还保留了抗原性，从而实现了抗体标记的多样化组合。此外，该方法还允许其他荧光技术，如TUNEL和乙炔基脱氧尿苷（EdU）掺入测定。可以定性和定量地评估由共聚焦成像产生的图像，以分析各种参数，例如细胞类型的分布和相互关系。该技术简单、稳定、重现性好，可在100天内完成。
High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cellsin situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in ∼11 d.