Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture.
Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture.
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分离培养物中原代小鼠海马神经元磷酸肌醇的免疫荧光染色
DOI：
10.1016/j.xpro.2022.101549
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发表时间：
2022-09-16
期刊：
影响因子：
--
通讯作者：
Liu, Jia-Jia
中科院分区：
文献类型：
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作者：
Guo, Zhenzhen;Tong, Chunfang;Yang, Yanrui;Liu, Jia-Jia
磷酸肌醇 (PIP) 是一种低丰度的膜脂，在细胞生理学中具有至关重要的功能。为了研究它们在神经元中的亚细胞分布，我们优化了使用市售抗体对细胞内或质膜 PIP 进行免疫荧光染色的方案。在这里，我们描述了分离培养物中原代小鼠海马神经元的制备和转染，然后进行免疫荧光染色和 PIP 信号的定量分析。此外，我们将该协议的应用扩展到位于细胞膜细胞质小叶的蛋白质。有关该协议的使用和执行的完整详细信息，请参阅。用于检测培养小鼠神经元中膜脂质的方案 培养神经元以分析脂质的亚细胞分布 质膜和内膜中脂质的差异免疫染色 该方案应用于膜细胞质小叶的蛋白质 出版商注：进行任何实验方案都需要遵守当地实验室安全和道德机构指南。磷酸肌醇 (PIP) 是一种低丰度的膜脂，在细胞生理学中具有至关重要的功能。为了研究它们在神经元中的亚细胞分布，我们优化了使用市售抗体对细胞内或质膜 PIP 进行免疫荧光染色的方案。在这里，我们描述了分离培养物中原代小鼠海马神经元的制备和转染，然后进行免疫荧光染色和 PIP 信号的定量分析。此外，我们将该协议的应用扩展到位于细胞膜细胞质小叶的蛋白质。
Phosphoinositides (PIPs) are low-abundant membrane lipids with critically important functions in cellular physiology. To investigate their subcellular distribution in neurons, we optimized protocols for immunofluorescence staining of intracellular or plasma membrane PIPs with commercially available antibodies. Here, we describe the preparation and transfection of primary mouse hippocampal neurons in dissociated culture, followed by immunofluorescence staining and quantitative analysis of PIP signals. In addition, we expand the application of the protocol to proteins located at the cytoplasmic leaflet of cellular membranes. For complete details on the use and execution of this protocol, please refer to. A protocol for detecting membrane lipids in cultured mouse neurons Culturing of neurons for analysis of the subcellular distribution of lipids Differential immunostaining of lipids in the plasma membrane and endomembranes Application of the protocol to proteins at the cytoplasmic leaflet of membranes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Phosphoinositides (PIPs) are low-abundant membrane lipids with critically important functions in cellular physiology. To investigate their subcellular distribution in neurons, we optimized protocols for immunofluorescence staining of intracellular or plasma membrane PIPs with commercially available antibodies. Here, we describe the preparation and transfection of primary mouse hippocampal neurons in dissociated culture, followed by immunofluorescence staining and quantitative analysis of PIP signals. In addition, we expand the application of the protocol to proteins located at the cytoplasmic leaflet of cellular membranes.