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首页> 外文期刊>The European Journal of Neuroscience >Fluorescence-activated sorting of fixed nuclei: A general method for studying nuclei from specific cell populations that preserves post-translational modifications
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Fluorescence-activated sorting of fixed nuclei: A general method for studying nuclei from specific cell populations that preserves post-translational modifications

机译：固定核的荧光激活分选：研究特定细胞群中保留翻译后修饰的核的一般方法

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摘要

Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.
机译：构成学习和记忆基础的持久性大脑变化是由突触活动触发的。活动如何对神经元产生长期影响是神经科学中的一个主要问题。在这种情况下，从细胞质到细胞核的信号通路以及转录和表观遗传修饰的最终变化特别重要。然而，他们研究的主要困难来自脑组织的细胞异质性。一种有前途的方法是直接纯化已鉴定的核。我们已经使用小鼠纹状体开发了一种快速有效的方法，用于从固定的成年大脑中分离细胞类型特异性核（荧光激活的固定核排序; FAST-FIN）。快速给动物灌输甲醛固定剂，该固定剂可停止酶促反应并将组织维持在死亡时的状态，包括可溶性蛋白（如GFP）的核定位以及细胞类型之间的核大小差异。随后将组织用Dounce匀浆器解离，并通过碘克沙醇密度梯度离心制备的细胞核。然后可以用特异性抗体对纯化的固定核进行免疫染色，并通过流式细胞仪进行分析或分选。简单的标准可以区分神经元和非神经元细胞。表达荧光蛋白的免疫标记和转基因小鼠可用于鉴定特定的细胞群，并且可以有效地分离出这些群的细胞核，甚至罕见的细胞类型，如表达小白蛋白的中间神经元。 FAST-FIN可以保存和研究动态和不稳定的翻译后蛋白质修饰。它应适用于其他组织和物种，并允许研究DNA及其修饰。

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