| Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System
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Author:
Date:
2017-10-20
[Abstract] A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al., 2012; Ramirez et al., 2013). Labeling and stimulating those neurons will activate the memory trace (Liu et al., 2012; Ramirez et al., 2013). Memory appears to be spread over different regions of the brain rather than being localized to one area. Therefore, the methods used to trace memory have the ability to improve our understanding of neuronal circuits. In this protocol, we introduce a doxycycline-inducible expression system to label the specific neurons associated with the original memory trace.
[摘要] 存储器跟踪（也称为存储器枚举）被理论化为大脑中物理存储器存储的机制（Silva等人，2009; Josselyn，2010），并且内存跟踪与一个 特定的神经元群体（Liu et al。，2012; Ramirez等人，2013）。 标记和刺激那些神经元将激活记忆痕迹（Liu et al。，2012; Ramirez等人，2013）。 记忆似乎分布在大脑的不同区域，而不是局限于一个区域。 因此，用于跟踪记忆的方法有能力提高我们对神经元电路的理解。 在本协议中，我们引入多西环素诱导表达系统来标记与原始记忆痕迹相关的特定神经元。
【背景】记忆痕迹是记忆被存储为大脑物理或生物化学变化的理论手段（Ryan等人，2015）。在二十世纪初德国动物学家理查德·塞蒙（Richard Semon）制定记忆追踪概念之后，记忆存储的具体过程一直是神经科学领域辩论的一个未解决的话题（Poo et al。，2016）。尽管记忆机制已经成为几十年来的争论焦点，但已经一致认为，特定的神经元被用于记忆的存储（Liu等人，2012; Ramirez等人， ...
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| Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
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Author:
Date:
2016-11-20
[Abstract] Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal ...
[摘要] 专业吞噬细胞通过吞噬作用内化自身和非自身颗粒以启动先天免疫应答。内化后，形成的吞噬体通过与内体和溶酶体的融合和裂变事件成熟，以获得更酸性，氧化和水解的环境用于其货物的降解。有趣的是，吞噬体成熟动力学在细胞类型和细胞活化状态之间不同。该协议允许使用流式细胞术定量不同类型的吞噬细胞中单个细胞器水平上的吞噬体成熟动力学。在这里，卵白蛋白（OVA）耦合的颗粒用作吞噬作用模型系统在树突状细胞（DC），其通过吞噬内化。在不同的时间点之后，吞噬体成熟参数，例如OVA的吞噬体降解和溶酶体蛋白（例如LAMP-1）的获得，可以通过流式细胞器细胞计数以高度定量的方式同时测量。这些读出可以与其他吞噬体功能相关，例如抗原降解，在DC中的加工和负载。
[背景] ...
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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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