| Imaging Thick Lymph Node Tissue Sections
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Author:
Date:
2016-09-20
[Abstract] Our protocol describes a simple procedure for imaging thick lymph node sections by 2-photon microscopy. Lymph nodes are sectioned using a vibratome (vibrating microtome) to produce slices of tissue that can then be stained with fluorescently labeled antibodies. The thick tissue sections (150-200 μm depth) allow for the detection of cell clustering that is typically under-represented in thin sections (10-20 μm) used for conventional confocal microscopy. Application of 2-photon microscopy facilitates imaging through the thick volume of the vibratome sections. In combination with automated image processing software, a thick lymph node cross-section image also facilitates quantitation of cellular events within a relatively large area of the tissue, thus providing a clearer picture on the ...
[摘要] 我们的协议描述了一个简单的程序，通过双光子显微镜成像厚淋巴结切片。 使用vibratome（振动切片机）切割淋巴结以产生组织切片，然后可用荧光标记的抗体染色。 厚组织切片（150-200μm深度）允许检测细胞聚集，其通常在用于常规共聚焦显微镜的薄切片（10-20μm）中表现不足。 双光子显微镜的应用有利于成像通过厚的体积的vibratome部分。 与自动图像处理软件组合，厚的淋巴结横截面图像还有助于在组织的相对大的区域内的细胞事件的定量，从而提供关于感兴趣的细胞事件的空间分布的更清晰的图像（例如 。，T细胞聚类）。 该方法也可以容易地应用于其它组织，例如脾或皮肤。
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| Whole-mount Enteroid Proliferation Staining
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Author:
Date:
2016-06-20
[Abstract] Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids ...
[摘要] 小肠类器官，也称为肠袢，已经成为越来越多地用于肠道生物学的体外模型，因为它们重现了在肠隐窝内的各种上皮细胞（Mahe等人 ，2013; Sato et al。，2009）。在这些培养物中评估生长动力学是理解基因表达的改变，用保护性和毒性试剂处理以及遗传突变改变隐窝生长和存活所必需的性质以及细胞内单个细胞的干细胞性质的重要步骤地穴。该方案描述了在隐窝内三维的增殖细胞的可视化方法（Barrett等人，2015）。使用EdU掺入的肠衣的整体增殖染色使得研究者能够观察到肠内的所有增殖细胞，而不是如在包埋和切片中所见到的在薄切片中获得生长信息，确保来自干细胞区室的增殖的真实表现到隐窝的终末分化细胞。
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| Single Molecule RNA FISH in the Mammalian Oocyte
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Author:
Date:
2015-12-05
[Abstract] RNA fluorescence in situ hybridization is a method to localize and measure gene expression in individual cell or tissue. Using multiple specific fluorescently labeled oligonucleotides greatly increases signal-to-noise ratio and thus enables detection of single RNA molecule. Around forty different DNA oligonucleotides designed to common RNA target and labeled with single fluorophore at 3´ terminus hybridizes with target RNA in fixed cells. We adapt this method to visualize target RNA in the mammalian oocyte. The ability to detect single transcript in the mammalian oocyte was challenging due to its large cell size. This method consists of four simple steps: fixation, permeabilization, hybridization and imaging. The protocol is adapted to this large nonattached cell to visualize ...
[摘要] RNA荧光原位杂交是定位和测量个体细胞或组织中的基因表达的方法。 使用多个特异性荧光标记的寡核苷酸大大增加信噪比，从而使得能够检测单个RNA分子。 大约四十个不同的DNA寡核苷酸设计为常见的RNA靶标，并在3'端用单个荧光团标记，与固定细胞中的靶RNA杂交。 我们适应这种方法可视化目标RNA在哺乳动物卵母细胞。 在哺乳动物卵母细胞中检测单个转录物的能力由于其大的细胞大小而具有挑战性。 该方法由四个简单的步骤组成：固定，预稳定化，杂交和成像。 该方案适应这种大的非附着细胞以显现母体RNA。
各种荧光团的组合允许检测更多的RNA靶标。 该方法可以与细胞器标记一起使用或用免疫荧光方案扩增。
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