这项工作得到了研究院 (R01GM11755) 对 P.W.J. 的支持, 以及由国家癌症研究所 (NIH) (CA009110) 向 S.R.W. 和 j.h 提供的培训补助金奖学金。

约翰·霍普金斯大学机构动物保育和使用委员会批准了对老鼠的使用。利用半同步第一波精子发生的方法, 对小鼠 (20-26 天产后、民进党) C57BL/6J 进行了实验。然而, 这种技术也可以使用成年小鼠进行。
1. 小鼠输精管解剖和分离
<ol><li>准备表 1和表 2中描述的修复/溶解解决方案和抗体稀释缓冲 (ADB)。</li>
	<li>准备一个35毫米的培养皿, 3 毫升的1x 磷酸缓冲盐水 (1x PBS, pH 7.4), 和另一个35毫米的菜, 每只老鼠2毫升的固定/溶解溶液。</li>
	<li>通过颈部脱位或 CO2窒息, 用70% 乙醇喷洒腹腹部。用无菌剪刀打开盆腔, 形成 V 形开口。然后用镊子拔出附睾脂肪垫, 避免扰乱白。将睾丸放在含有 1x PBS 的35毫米培养皿中。 注意:利用第一波的精子发生, 以观察丰富的细胞人口的利益14。在不同的小鼠年龄 (表 3) 丰富了精子发生的特定阶段。</li>
	<li>用锋利的尖刺钳刺穿组织, 取出睾丸的白, 并收集松散的精小管。将小管转移到新的35毫米培养皿中, 其中含有2毫升的固定/溶解液。</li>
	<li>在室温下孵育5分钟的固定/溶解液中的小管。</li>
</ol>2. 生精小管的制备
<ol><li>通过用液体阻挡笔勾勒出幻灯片的边缘, 准备一张多聚 l-赖氨酸涂层玻璃玻片。</li>
	<li>将100µL 的固定/溶解液应用于玻璃滑板的中心。</li>
	<li>用无菌钳和剪刀轻轻地把生精小管分开。切割长的单个小管段约20毫米的长度。 注意:为了可视化对长时间固定暴露敏感的结构, 如体, 在 1x PBS 中先分离小管, 然后直接将在100µL 的聚赖氨酸片表面上的小管剁碎。</li>
	<li>将五 20 mm 长的生精小管段转移到含有固定/溶解溶液的制备的多聚 l-赖氨酸涂层玻璃玻片上。</li>
	<li>用无菌剪刀把生精小管分成1.5 到3.0 毫米的部分。</li>
	<li>用无菌钳将小管段排列在玻片上, 使无组织重叠, 小管分布均匀。 注意:在玻片上的小管段的最佳数目是在20和40之间。</li>
</ol>3. 生精小管的挤压
<ol><li>将含有分散的生精小管段的玻璃玻片转移到台式上。使用实验室擦拭去除多余的液体, 避免在壁球过程中丢失组织。</li>
	<li>将片 (22 x 60-1.5 mm) 涂抹在玻璃片的顶部, 用手掌的脚跟按压 10-20 秒。从生精小管中应用足够的力来分散精是很重要的。 注意:用解剖显微镜观察小管, 以优化挤压步骤, 使所有的管节都受到破坏。</li>
	<li>使用滑动钳, 立即闪光冻结玻璃在一个小杜瓦瓶的液体 N2 15 秒, 或直到液体停止起泡。如果 immunolabeling 立即, 删除片与直刃刀片, 罚款尖端钳或21口径针。 注意:为了获得最佳结果, 请立即 immunolabel 幻灯片 (请参见步骤 4)。然而, 在这一点上, 幻灯片可以保存在-80 ° c 最多两周。为了最大限度地延长蛋白质和细胞结构的寿命, 立即将幻灯片存储在干冰上或转移到-80 ° c 的冰箱, 并将片放在幻灯片上。当 immunolabeling 存储幻灯片时, 如步骤3.3 所述, 将幻灯片浸泡在液态 N2中15秒, 以删除片。</li>
</ol>4. Immunolabeling 的生精小管
<ol><li>在 1x pbs 中浸泡幻灯片, 并在包含 1x pbs 的50毫升 Coplin 罐中洗涤三次, 5 分钟。 注意:不要让幻灯片完全干燥在 immunolabeling。</li>
	<li>将1毫升的抗体稀释缓冲液涂抹在玻片上, 在湿化室中对 1-2 h 进行阻挡。</li>
	<li>去除抗体稀释缓冲液, 在湿化室中应用抗体稀释缓冲液中稀释的原抗体100µL。 注意:为取得最佳效果, 在4° c 的夜间孵育主抗体。使用较小数量的抗体 (例如50 µL), 用片或膜覆盖该幻灯片。验证和优化主抗体15。</li>
	<li>冲洗幻灯片三次5分钟, 在50毫升 Coplin 罐含有 1x PBS。 注意:为了增强洗涤, 使用小的磁性搅拌棒, 在低速的磁性搅拌板上搅拌 Coplin 罐, 或在低速时将 Coplin 罐放在台式搅拌机上。</li>
	<li>在室温下, 在湿化室内应用100µL 抗体稀释缓冲液中的二次抗体稀释1至 1.5 h。在一个黑暗的盒子中孵育幻灯片, 以避免荧光共轭抗体的照片漂白。 注意:使用较小数量的抗体 (例如50 µL), 用片或膜覆盖该幻灯片。</li>
	<li>在含有 1x PBS 的50毫升 Coplin 罐中, 冲洗两次幻灯片5分钟。</li>
	<li>将幻灯片装入包含 4 "、6-diamidino-2-吲 (DAPI、1.5 µg/mL) 的安装介质中, 并应用片 (22 x 60-1.5 mm)。</li>
	<li>用清晰的指甲油将片密封在幻灯片上, 以保存幻灯片, 防止片移动。</li>
</ol>5. 分析和成像小管壁球准备
<ol><li>使用荧光显微镜与自动化阶段, 使精确的 Z 轴运动和高分辨率相机的图像捕捉。或者, 使用激光共聚焦显微镜。 注意:在此步骤中使用任何可用的硬件和软件。例如, 在图 1和图 2中显示的图像是使用蔡司单元观察器 Z1 链接到一个虎鲸-闪光 4.0 CMOS 相机, 并与蔡司禅宗2012蓝色版图像软件进行分析。</li>
	<li>使用20X 目标评估幻灯片, 以确定壁球准备的质量。质量好的幻灯片具有均匀分布的单层原子核。 注意:幻灯片的某些部分可能比其他章节更好, 因此可以在使用更高的放大倍数的情况下, 找到幻灯片的最佳区域的坐标, 以便进一步分析。</li>
	<li>使用更高的放大率目标 (例如63X 或 100X), 捕获具有一致单层核的幻灯片区域。选定区域后, 设置上部和下部 Z 堆栈, 以确保捕获所有聚焦光。 注意:在上限和下限之间捕获的 Z 栈的最优数通常由图像采集软件指定, 对每个目标都是不同的。</li>
	<li>使用图像处理软件来编译一个扩展的深度聚焦图像。该软件结合了聚焦光从每个 Z 栈, 是必不可少的最佳成像小管壁的准备工作。</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+in+the+immature+mouse+proceeds+faster+than+in+the+adult.">Spermatogenesis in the immature mouse proceeds faster than in the adult.</a> Int J Androl. 5 (3), 282-294 (1982).</li><li>Rodriguez, I., Ody, C., Araki, K., Garcia, I., Vassalli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+and+massive+wave+of+germinal+cell+apoptosis+is+required+for+the+development+of+functional+spermatogenesis.">An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.</a> EMBO J. 16 (9), 2262-2270 (1997).</li><li>Kangasniemi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+basal+and+FSH+dependent+cyclic+AMP+production+in+rat+seminiferous+tubules+staged+by+an+improved+transillumination+technique.">Modulation of basal and FSH dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique.</a> Anat Rec. 227 (1), 62-76 (1990).</li><li>Martianov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+arrest+of+spermiogenesis+and+germ+cell+apoptosis+in+mice+lacking+the+TBP-like+TLF/TRF2+gene.">Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene.</a> Mol Cell. 7 (3), 509-515 (2001).</li><li>Henriks&#233;n, K., Parvinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+apoptosis+of+male+germ+cells+in+the+rat:+Mechanisms+of+cell+death+studied+by+supravital+squash+preparations.">Stage-specific apoptosis of male germ cells in the rat: Mechanisms of cell death studied by supravital squash preparations.</a> Tissue Cell. 30 (6), 692-701 (1998).</li></ol>

作者没有什么可透露的。

小鼠已被证明是一个有用的模型有机体研究的细胞事件, 控制减数分裂进程的精子发生。此外, 有必要开发用于研究精子发生的工具, 因为许多事件, 如减数分裂前期 I 的退出, 都是性二。本协议描述了一种用于小鼠精子发生不同阶段可视化和研究的精曲管壁法。这种方法保留细胞完整性, 从而允许详细分析核和细胞质结构。本研究的代表性结果表明该协议在评估初级精减数分裂进程中的效用。此外,...

减数分裂是一个复杂的细胞事件, 其中一个单一的一轮 DNA 复制后, 连续两轮细胞分裂。在减数分裂的初始阶段, 必须协调几个减数分裂的特定事件, 以确保准确的染色体分离。这些事件包括完成同源重组, 在第一次减数分裂的姐妹 kinetochores 的共同取向, 和逐步失去内聚力配合, 以解决交叉之间的同系物。对这些过程进行精确的调控是保持生育力和防止染色体 missegregation 事件的必要条件, 这可能导致遗传发育障碍和自发性流产1。
虽然减数分裂的关键事件发生在男性和女性, 重要的时间和机械差异之间存在精子生成和卵子2。例如, 在女性减数分裂期间, 我发生在胚胎发育和 dictyate 阶段的逮捕, 直到青春期。相比之下, 精子发生在青春期就开始了, 在成年生活中也没有被逮捕。男性和女性减数分裂的区别强调需要制定方法, 专门用于评估这些过程在精和卵母细胞。目前, 评估减数分裂的进展主要依赖于使用染色质传播3,4,5。虽然染色质传播是有用的研究减数分裂染色体, 他们不能保持细胞完整性, 防止评估细胞结构, 如纺锤微管, 体, 核包络, 和端粒附着。活体成像和长期培养技术大大提高了我们对女性减数分裂的认识;然而, 类似的方法来可视化整个完整的细胞, 不太经常实施的研究的精子发生6,7。为了在整个雄性减数分裂中可视化动态事件, 我们已经适应了已建立的小管壁技术, 以快速评估开发鼠标精8,9的细胞学特征。这里描述的方法保持细胞的完整性, 使研究多细胞结构在不同阶段的精子发生。
这种小管壁技术是一种完整的细胞方法, 可以通过免疫荧光显微镜对细胞结构进行评估。常见的组织学方法, 以可视化的雄性小鼠减数分裂进展, 如苏木和曙红染色石蜡嵌入睾丸, 和荧光标记 cryosections 允许广泛的概述减数分裂进展。但是, 这些技术无法将单个单元格解析为对整个减数分裂1011中发生的事件进行详细分析所必需的程度。可视化减数分裂过程的替代技术依赖于对精的重大 chemiosmotic 中断来隔离和修复核材料3,4,5。这些化学处理阻碍了除原精以外的细胞类型的观察。Namekawa 最近描述的一种方法使研究界能够保护孤立精的核结构, 但需要使用一些实验室4可能不容易获得的 cytospin 和附件。相比之下, 小管壁技术只需要在大多数细胞生物学实验室中通常是标准的设备。
这里描述的小管壁方法可以用来可视化在精曲管内发现的各种细胞类型, 包括支持细胞, 精, 初级和次级精, 和精子。通过将这一技术与幼鼠精子发生的近同步第一波结合起来, 在通过减数分裂12的过程中, 可以获得生精细胞的富集种群。这一过程允许详细分析整个精子发生过程, 如早期前期事件, G2/MI 和中期到后期过渡, 和精子。此外, 小管壁的准备可用于可视化的细胞学特征的染色体 (如 interchromatid 域 (服务) 和 kinetochores) 和体 (中心和 pericentriolar 材料/矩阵)。壁球法可以很容易地与其他实验方法, 如染色质传播和蛋白质提取并行执行。此外, 该技术已成功地修改, 以存放活体生精细胞的幻灯片直接可视化13。
本文所述方法涉及一种全细胞精管壁球技术, 用于分析野生型 C57BL/6J 小鼠的 G2/MI 转变。用免疫荧光显微镜观察了初级精进入第一减数分裂的细胞学特征, 以观测减数分裂纺锤体。这种多才多艺的技术可以很容易地修改, 以可视化其他减数分裂阶段和不同的细胞类型。该技术还可用于替代的可视化策略, 如 DNA 和 RNA 鱼的方法。

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			<td style="width:137px;">N/A</td>
			<td style="width:167px;">1 in 1000</td>
		</tr>
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</table>

a seminiferous tubule squash technique for the cytological analysis of spermatogenesis using the mouse model

雄性的减数分裂是一个过程, 需要一些高度调控的细胞事件的协同作用。减数分裂过程中发生的错误会导致不孕、流产或遗传缺陷。从青春期开始到成年, 持续的半同步波精经历精子发生, 最终形成单倍体精子。第一波的小鼠精进行减数分裂开始出现在10天产后 (10 民进党), 并被释放到管腔的精管作为成熟精子35民进党。因此, 利用小鼠在这个发展的时间窗口, 以获得高度丰富的利益群体是有利的。在老年小鼠中, 由于连续生精波的作用, 对稀有细胞阶段的分析比较困难, 从而增加了微管内细胞数量的多样性。本文所描述的方法是一种易于实现的技术, 用于细胞学评价小鼠的精管中发现的细胞, 包括精, 精, 和精子。小管壁技术保持了孤立的雄性生殖细胞的完整性, 并允许检查不容易与其他技术可视化的细胞结构。为了证明这种小管壁技术的可能应用, 在精过程中, 通过前期到中期 I 过渡 (G2/MI 过渡), 对主轴总成进行了监测。此外, 中心复制, 减数分裂性染色体失活 (MSCI) 和染色体花束形成被评估为例子的细胞学结构, 可以观察使用这种小管壁法。这种技术可以用来精确地确定精子发生过程中的特定缺陷, 这是由突变或外源性扰动引起的, 从而有助于我们对精子生成的分子理解。

在这里, 我们使用小管壁的方法, 以可视化的过渡细胞人口经历前期到中期 I (G2/MI) 转变, 这是丰富的收获睾丸从幼鼠的第一波精子发生 (24民进党)。图 1描述了可以使用小管壁方法可视化的各种细胞阶段的代表性图像。中期 I 精的富集种群是使用抗α-蛋白 (图 1A) 的抗体可视化的。我们利用标记的早期和晚期减数分裂 I 阶段减数分...

Watch this Scientific Journal Video about A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model at JoVE.com

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

这个小管壁技术的目的是快速评估发育中鼠精的细胞学特征, 同时保持细胞完整性。这种方法允许研究精子发生的所有阶段, 并可以很容易地实现与其他生物化学和分子生物学方法的研究小鼠减数分裂。

用小鼠模型对精子发生细胞学分析的精管壁球技术

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

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14.8K 次观看.  Johns Hopkins Bloomberg School of Public Health. 这个小管壁技术的目的是快速评估发育中鼠精的细胞学特征, 同时保持细胞完整性。这种方法允许研究精子发生的所有阶段, 并可以很容易地实现与其他生物化学和分子生物学方法的研究小鼠减数分裂。

视频: A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

小鼠胚胎跖骨文化:软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

研究大鼠固定性诱导膝关节弯曲收缩的微型侵入性内固定技术

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

造血干细胞代谢分析

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.