| A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
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Author:
Date:
2021-04-05
[Abstract] The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis (Chang et al., 2020). This protocol was used in the 3D ...
[摘要] [摘要]自由生活的线虫秀丽隐杆线虫是研究发育生物学的流行模型系统。在这里，我们描述了详细的协议，以高压冷冻线虫的胚胎（解剖后离体，或完整的蠕虫内），然后快速冷冻替代。经过处理的样品适合通过常规电子显微镜（EM）或更新的体积EM（vEM）方法（如Focuse d离子束扫描电子显微镜（FIB-SEM））进行超微结构分析。的细胞特征，例如超微结构的NUCL耳信封，染色体，内质网和线粒体保存良好这些实验程序后，并产生精确的三维模型用于可视化和分析（张等人，2020）。在秀丽隐杆线虫合子的前核相遇后，该方案被用于膜和染色体的3D重建（Rahman等，2020）。
[背景技术]线虫是自由生活线虫具有许多特性，使其适合于科学的研究：（1）将蠕虫是〜1毫米长; ...
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| In vitro Time-lapse Imaging of Primary Cilium in Migrating Neuroblasts
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Author:
Date:
2020-11-20
[Abstract] Neuronal migration is a critical step for the development of neuronal circuits in the brain. Immature new neurons (neuroblasts) generated in the postnatal ventricular-subventricular zone (V-SVZ) show a remarkable potential to migrate for a long distance at a high speed in the postnatal mammalian brain, and are thus a powerful model to analyze the molecular and cellular mechanisms of neuronal migration. Here we describe a methodology for in vitro time-lapse imaging of the primary cilium and its related structures in migrating V-SVZ-derived neuroblasts using confocal or superresolution laser-scanning microscopy. The V-SVZ tissues are dissected from postnatal day 0-1 (P0-1) mouse brains and dissociated into single cells by trypsinization and gentle pipetting. These cells are then ...
[摘要] [摘要]神经元迁移是大脑中神经元回路发展的关键步骤。产后心室-脑室下区（V-SVZ）中产生的未成熟新神经元（神经母细胞）显示出巨大的潜力，可以在产后哺乳动物脑中高速长距离迁移，因此是分析分子和神经元的强大模型。神经元迁移的细胞机制。在这里，我们描述了一种使用共聚焦或超分辨率激光扫描显微镜对V-SVZ衍生的成神经细胞进行迁移的初级纤毛及其相关结构的体外延时成像方法。从出生后的第0-1天（P0-1）小鼠脑中解剖V-SVZ组织，并通过胰蛋白酶消化和温和的移液将其分离成单个细胞。然后用编码目的基因的质粒转导这些细胞，通过离心聚集，并在Matrigel中培养2天。通过共聚焦或超分辨率激光扫描显微镜获取培养的神经母细胞及其睫状结构（包括睫状膜和基体）迁移行为的时移图像。该方法提供了有关成神经细胞形态和睫状结构时空动态的信息，并且广泛适用于各种物种中各种类型的迁移神经元和非神经元细胞。
[背景]在出生后的大脑中，神经干细胞驻留在侧脑室侧壁内衬的心室-心室下区（V-SVZ）中，并不断生成未成熟的新神经元（神经母细胞）（Obernier和Alvarez-Buylla，2019）。这些成神经细胞形成链状细胞聚集体，并通过鼻尖迁移流（RMS）彼此迁移至嗅球（OB）（Luskin，1993; Lois and Alvarez-Buylla，1994; Lois et ...
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| Surgical Induction of Endometriosis in Female Mice
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Author:
Date:
2020-09-20
[Abstract] Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterine cavity. It is frequently associated with pain, infertility and a reduced quality of life, and it lacks adequate treatment. Several rodent models of endometriosis have been developed through heterologous and homologous transplantation of endometrial tissue into the abdominal compartment. Here we describe a surgical procedure to generate a syngeneic model of endometriosis in immunocompetent mice with intact uterine and ovarian tissues. In this model, four uterine fragments from a donor mouse at diestrus are sutured to the abdominal wall of a recipient mouse. One month after surgeries, endometrial implants develop into cysts with glandular epithelium and stroma, mimicking ...
[摘要] [摘要 ] 子宫内膜异位症是一种常见的妇科疾病，其特征是子宫腔外存在子宫内膜组织。它经常与疼痛，不育和生活质量下降有关，并且缺乏适当的治疗。子宫内膜异位症的几种啮齿动物模型是通过将子宫内膜组织异源和同源移植到腹腔中而开发的。在这里，我们描述了在具有完整子宫和卵巢组织的免疫功能小鼠中产生子宫内膜异位症的同基因模型的手术程序。在该模型中，将来自供体小鼠在二头肌的四个子宫碎片缝合到受体小鼠的腹壁上。手术一个月后，子宫内膜植入物发展成具有腺上皮和间质的囊肿，模仿了子宫内膜异位症女性中观察到的子宫内膜异位病变。因此，该小鼠模型为研究子宫内膜异位症的病理生理学和潜在治疗方法的有效性提供了有价值的工具。
[背景 ] 子宫内膜异位是由子宫外子宫内膜组织的生长所定义的一种慢性妇科疾病，主要在骨盆和腹部表面生长（Zondervan et al。，2018）。它影响了育龄妇女的10％，并且与疼痛，不孕症和生活质量下降有关（Fourquet 等人，2011;Márki 等人，2017; Zondervan 等人，2018）。子宫内膜异位症的可用药物和手术疗法具有不良作用，并且无法长期缓解症状（Falcone和Flyckt，2018年）。因此，需要新的治疗策略，其发展依赖于动物模型的建立，该模型概括了临床子宫内膜异位症的病理生理和行为特征。
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