Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.
Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.
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DOI：
10.1038/srep26013
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发表时间：
2016-05-16
影响因子：
4.6
通讯作者：
Lax NZ
中科院分区：
文献类型：
--
作者：
Phillips J;Laude A;Lightowlers R;Morris CM;Turnbull DM;Lax NZ
可重复性使免疫荧光标记和大体积组织的成像，以提供更好地了解细胞形态和不同细胞类型之间的空间相互作用之间的三维关系。在目前的研究中，我们优化了小鼠和人脑组织中神经元和线粒体蛋白的被动免疫荧光标记和免疫荧光标记，以进一步了解线粒体疾病中发生的神经变性机制。这是第一项利用多聚甲醛固定并冷冻保护的人小脑与福尔马林固定组织结合的研究，为使用存档组织开辟了进一步的途径。我们优化了水凝胶包埋和被动清除脂质从小鼠（n = 5）和人类（n = 9）小脑，以及开发一种免疫荧光协议，一贯标记不同的神经元结构域以及血管。除了可视化大型结构外，我们还能够可视化被动清除组织中的线粒体蛋白，以揭示与线粒体疾病相关的呼吸链缺陷。我们还通过剥离抗体和重新探测小脑来证明组织的多种用途。该技术允许询问大量完整的脑样本，以更好地了解线粒体疾病中发生的复杂病理变化。
CLARITY enables immunofluorescent labelling and imaging of large volumes of tissue to provide a better insight into the three dimensional relationship between cellular morphology and spatial interactions between different cell types. In the current study, we optimise passive CLARITY and immunofluorescent labelling of neurons and mitochondrial proteins in mouse and human brain tissues to gain further insights into mechanisms of neurodegeneration occurring in mitochondrial disease. This is the first study to utilise human cerebellum fixed in paraformaldehyde and cryoprotected in conjunction with formalin-fixed tissues opening up further avenues for use of archived tissue. We optimised hydrogel-embedding and passive clearance of lipids from both mouse (n = 5) and human (n = 9) cerebellum as well as developing an immunofluorescent protocol that consistently labels different neuronal domains as well as blood vessels. In addition to visualising large structures, we were able to visualise mitochondrial proteins in passively cleared tissues to reveal respiratory chain deficiency associated with mitochondrial disease. We also demonstrate multiple use of tissues by stripping antibodies and re-probing the cerebellum. This technique allows interrogation of large volumes intact brain samples for better understanding of the complex pathological changes taking place in mitochondrial disease.