Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment.
Improved Methods for Fluorescence Microscopy Detection of Macromolecules at the Axon Initial Segment.
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DOI：
10.3389/fncel.2016.00005
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发表时间：
2016
影响因子：
5.3
通讯作者：
Laezza F
中科院分区：
文献类型：
--
作者：
Alshammari MA;Alshammari TK;Laezza F
轴突起始段（AIS）是神经元动作电位起始所需的亚细胞区室。AIS簇中的支架和调节蛋白与离子通道一起确保电信号的完整性。干扰这种蛋白质网络的结构会对神经元的极性、兴奋性、细胞与细胞的连接性和脑回路的可塑性产生深远的影响。因此，精确可视化AIS组件的能力为解析神经元功能的关键分子决定因素提供了宝贵的机会。基于荧光的免疫标记是用于神经元中精细结构的形态学和分子表征的灵敏方法。然而，即使与共聚焦显微镜结合，免疫荧光检测AIS元件也受到固定材料引起的抗原性丧失的限制。这一技术障碍对单独检测AIS组分或与其他标志物组合检测AIS组分造成了重大限制。在这里，我们设计了改进的方案，靶向共聚焦免疫荧光检测AIS标志物成纤维细胞生长因子14（FGF 14）与细胞凋亡相关蛋白Anklym-G，支架蛋白β IV-血影蛋白，电压门控Na+（Nav）通道（特别是Nav1.6亚型）和小鼠大脑中关键细胞类型特异性神经元标志物，如小清蛋白，钙结合蛋白和NeuN。值得注意的是，我们证明了用含有1%甲醛和0.5%甲醇的市售溶液对动物进行心内灌注，然后用冷丙酮短暂固定是用于FGF 14和其他AIS标志物检测的最佳和灵敏的方案，其保证了优异的组织完整性。随着程序的变化，我们还显着提高了Nav1.6的检测，Nav1.6是一种以其固定剂敏感性而闻名的Nav亚型。总的来说，这项研究提供了一套免疫组化方法，可以在保存良好的组织结构中对不可见的分子进行良好的染色。在改善AIS生理学和细胞生物学的具体研究的同时，我们的全面研究也可以作为优化其他固定剂敏感蛋白质免疫检测的路线图，扩大大脑研究的方法库。
The axonal initial segment (AIS) is the subcellular compartment required for initiation of the action potential in neurons. Scaffolding and regulatory proteins at the AIS cluster with ion channels ensuring the integrity of electrical signaling. Interference with the configuration of this protein network can lead to profound effects on neuronal polarity, excitability, cell-to-cell connectivity and brain circuit plasticity. As such, the ability to visualize AIS components with precision provides an invaluable opportunity for parsing out key molecular determinants of neuronal function. Fluorescence-based immunolabeling is a sensitive method for morphological and molecular characterization of fine structures in neurons. Yet, even when combined with confocal microscopy, detection of AIS elements with immunofluorescence has been limited by the loss of antigenicity caused by fixative materials. This technical barrier has posed significant limitations in detecting AIS components alone or in combination with other markers. Here, we designed improved protocols targeted to confocal immunofluorescence detection of the AIS marker fibroblast growth factor 14 (FGF14) in combination with the cytoskeletal-associated protein Ankyrin-G, the scaffolding protein βIV-spectrin, voltage-gated Na+ (Nav) channels (especially the Nav1.6 isoform) and critical cell type-specific neuronal markers such as parvalbumin, calbindin, and NeuN in the mouse brain. Notably, we demonstrate that intracardiac perfusion of animals with a commercially available solution containing 1% formaldehyde and 0.5% methanol, followed by brief fixation with cold acetone is an optimal and sensitive protocol for FGF14 and other AIS marker detection that guarantees excellent tissue integrity. With variations in the procedure, we also significantly improved the detection of Nav1.6, a Nav isoform known for its fixative-sensitivity. Overall, this study provides an ensemble of immunohistochemical recipes that permit excellent staining of otherwise invisible molecules within well-preserved tissue architecture. While improving the specific investigation of AIS physiology and cell biology, our thorough study can also serve as a roadmap for optimizing immunodetection of other fixative-sensitive proteins expanding the repertoire of enabling methods for brain studies.