| Establishing an Adult Mouse Brain Hippocampal Organotypic Slice Culture System that Allows for Tracing and Pharmacological Manipulation of ex vivo Neurogenesis
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Author:
Date:
2021-01-05
[Abstract] The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the ...
[摘要] [摘要]海马的功能取决于成年海马神经发生的过程，该过程是这种结构异常的神经发育的基础，并且在中枢神经系统病理中也经常受到影响。因此，对该过程的操纵代表了重要的治疗目标。为了确定潜在的策略，器官型成人大脑切片正在成为一种有价值的工具。近年来，此方法已得到完善，在此我们提出一种组合协议， 汇集了这些改进，以实现成人海马切片的长期培养。我们采用了一种切片技术，可将必要的传入输入保留在海马上以及无血清培养条件下，因此可以延长培养时间。为了维持切片中的神经源性潜力，我们利用神经胶质生成抑制剂吲哚美辛。使用EdU保留分析使我们能够评估药理干预对神经发生的影响。通过这些改进，我们建立了一种简单可靠的方法来研究小分子/药物对离体增殖和神经元形成的影响，这将有助于未来发现驱动的药物筛选。
[背景技术]海马是具有高度的可塑性作为整个生命齿状回中正在进行的神经发生的结果，脑的独特区域。成年海马神经发生的这一过程始于在亚颗粒区（SGZ）中神经干细胞（NSC）的不对称分裂，该过程保留了干细胞池并生成了准备用于神经元分化的祖细胞（Kempermann等人，2004；Anacker和Hen ，2017； ...
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| Quantitative Kinetic Analyses of Histone Turnover Using Imaging and Flow Cytometry
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Author:
Date:
2020-09-05
[Abstract] Dynamic histone changes occur as a central part of chromatin regulation. Deposition of histone variants and post-translational modifications of histones are strongly associated with properties of chromatin status. Characterizing the kinetics of histone variants allows important insights into transcription regulation, chromatin maintenance and other chromatin properties. Here we provide a protocol of quantitative and sensitive approaches to test the timing of incorporation and dissociation of histones using a two-color SNAP-labeling system, labelling pre-existing and newly-incorporated histones distinctly. Together with cell cycle synchronization methods and cell cycle markers, this approach enables a pulse-chase analysis to determine the turnover of histone variants during the cell cycle, ...
[摘要] [摘要] 动态的组蛋白变化是染色质调节的核心部分。组蛋白变体的沉积和组蛋白的翻译后修饰与染色质状态的属性密切相关。表征组蛋白变体的动力学特性可为深入了解转录调控，染色质维持和其他染色质特性提供重要信息。在这里，我们提供了一种定量和敏感方法的协议，以使用双色SNAP标记系统测试组蛋白的结合和解离时间，分别标记预先存在的和新结合的组蛋白。结合细胞周期同步方法和细胞周期标志物，这种方法可以进行脉冲追踪分析，以确定在细胞周期内使用成像或流式细胞仪方法以单细胞分辨率检测到的组蛋白变体的周转率。除了测试整体组蛋白更新，还可以使用成像方法解决组蛋白变体的细胞周期依赖性细胞定位。
[背景] 染色质重塑是真核细胞众多基本细胞活动的一部分（Geiman 和Robertson，2002；Clapier 和Cairns ，2009）。转录因子和RNA聚合酶的可及性通常与DNA甲基化和染色质状态的变化相关，包括可及性，翻译后的组蛋白修饰和组蛋白变体的沉积。组蛋白变体差异性地调节调节发育，细胞分化或其他生理活动的基因表达（Banaszynski 等，2010）。它们在DNA修复，端粒维护，异染色质形成和染色质分离中也发挥着不同的作用（Henikoff 和Smith，2015； Zink和Hake，2016）。此外，组蛋白变体的掺入失调与癌症有关（Vardabasso et ...
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| Immunofluorescent Staining of Mouse Intestinal Stem Cells
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Author:
Date:
2016-02-20
[Abstract] Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.
Figure 1. A schematic depicting a crypt-villus forming organoid, and visualization of Paneth cells by immunofluorescence staining. Left: Small intestinal organoids grow as crypt-villus structures that contain all of the ...
[摘要] 可以进行类器官的免疫荧光染色以显现细胞行为的分子标志物。例如，通过掺入核苷酸（EdU）标记的细胞增殖，或观察肠分化的标志物，包括paneth细胞，杯状细胞或肠细胞（参见图1）。在这个协议中，我们详细的方法来修复，透化，染色和安装肠组织，通过免疫荧光共聚焦显微镜分析。
图1.描绘隐窝 - 绒毛形成类器官的示意图，通过免疫荧光染色观察Paneth细胞。肠器官类生长为含有所有肠的多种分化谱系的隐窝 - 绒毛结构。右：免疫荧光染色可用于显现器官类型中的单个细胞类型。通过染色溶菌酶（"Lyso，"Green）显示paneth细胞，其显示位于隐窝碱基的Paneth细胞。 F-肌动蛋白（红色）显示在上皮的顶端表面的隐窝结构，DAPI（蓝色）揭示细胞核。比例尺为25μm。
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