本文NSERC格兰特＃299280，IRSST作为本科学生研究奖为M. CYR的奖学金（七）家长和FRSQ的支持。我们感谢执行上的视频记录的操作Yoan Lemieux Laneville。

<html>任何操作之前，你必须确定要使用取决于你进行的实验和仪器在您的性格筋组。对于我们而言，腹筋选择，因为他们都较小，因而更容易操纵时，测量的截面积和装修进入生物反应器室。 请注意，所有仪器高压灭菌或用70％乙醇消毒。此外，每个工作站旁放置一个喷雾瓶，含70％乙醇消毒每次操作前实验者手套。 第1部分：提取尾切除后，仔细地操纵其四肢，以避免损害组织。此外，保护细胞的活力，所有操作都进行了冷盐溶液。 1A）材料： <ul><li>冷盐溶液（D - PBS） </li><li>碎冰</li><li>表面保护</li><li>菜板</li><li>个人操纵板</li><li> 2 500毫升菜</li><li> 2 2L玻璃菜</li><li>胶带</li><li> 1镊子</li><li> 1钳</li><li> 1镊子站</li><li> 1对手术剪</li><li> 1手术刀</li><li> 1对手术剪 <img src="/files/ftp_upload/2176/2176fig1.jpg" alt="图1" /> 方向识别个人操纵（“P”，“近端” 图1。） </li></ul> 1B）工作站： <ol><li>摊开的表面保护和砧板放在上面。 </li><li>填写每两个较大的玻璃菜中途碎冰，并放置在纸张上。 </li><li>坚持一个磁带上的操纵板的一个角落的一块，并确定了一封信，表明近端。 </li><li>两个小玻璃中途菜肴和操纵板凹槽填充冷盐溶液。它们放置在含有冰的大玻璃菜。 </li><li>表面上保护组织的所有消毒。 </li><li>转移到尾部的玻璃含盐水菜之一。 </li></ol> 1C）操作： <ol><li>尾部近端的解剖观察，以区别于背侧腹侧。腹侧有一个数量较大的肌腱和血管，可轻轻挤压尾部产生微小的血液滴核实。 </li><li>使用的手术剪，切沿尾部背侧皮肤，如果你想提取的腹筋（或沿腹端，如果您需要背筋）。 </li><li>打开切口近端和删除通过精心操纵尾部两端使用产钳或手指和皮肤。 </li><li>在盐溶液的血液和头发用清水冲洗干净，并转移到剩余的玻璃盘包含新鲜的溶液试样。更换手套。 光显微镜下观察显示了一个横向的第6个肌腱组。一位来自各组肌腱是连接到每个椎体的尾部结束，因此，我们在椎间盘切揭露新的肌腱沿切割。 <img src="/files/ftp_upload/2176/2176fig2.jpg" alt="图2" /> 图2横向光学显微镜下观看了鼠尾节。 </li><li>穿过软骨2-3椎体手术剪短的末端。放在砧板上的尾巴，削减使用手术刀，然后立即在解决方案取代的软组织。 </li><li>使用镊子，从远端尾部拉出一个肌腱。 </li><li>在两端的温柔举行，放入个人操纵板的肌腱​​。 重复最后两个步骤，直到没有更多的筋可以梳理出切椎较短时，新的组织需要公开的。 </li></ol>第2部分：截面积测量7 当组织发生机械特性或刺激，其机械性能正常化内肌腱的力量，强调。这就是为什么我们评估的横截面面积。 2A）材料： <ul><li>光学测微计</li><li>体视显微镜</li><li>数码相机</li><li>边缘识别和简介重建算法5,6 </li><li>冷盐溶液（D - PBS） </li><li> 1 20 -200μL微量移液器</li><li> 1光纤微米调整的六角扳手</li><li> 2镊子 / files/ftp_upload/2176/2176fig3.jpg“ALT =”图3“/&gt; 图3光纤千分尺。 </li></ul> 2B）工作站： <ol><li>打开软件（数码相机和算法）。 </li><li>将光纤微米，以便有足够的空间来装载在仪器的肌腱。 </li><li>组织的工具和玻璃盘提取筋近在咫尺。 </li></ol> 2C）操作： <ol><li>冷盐溶液填充测量车厢，直到两个锚系统被淹没。 </li><li>拿着镊子的两端的肌腱转移到车厢。 </li><li>通过微量移液器吸，拉硅管在同一时间进入一个肢体。吸，直到最后超出了固定环延伸。 </li><li>拧紧固定环，压缩管，并安装在立体显微镜下仪器。 </li><li> 105X的放大倍率，同时观察组织，直到的折痕不再感知舒展筋。通过“额外的0.40％。 <img src="/files/ftp_upload/2176/2176fig4.jpg" alt="图4" /> 图4。投影105X的放大倍率和拉伸后肌腱。 </li><li>调整位置和重点，以保持一个清晰的图像超过180 °旋转肌腱。 </li><li>以每10 °旋转的照片，无需修改紧张，焦点或位置。仅使用旋转轴进行旋转。由于它的圆锥形，这些步骤可能会重复沿肌腱的不同点。 </li><li>激活边缘识别和轮廓重建算法，以分析您已注册的数据库。 </li><li>免费肌腱松开固定环和使用的硅微量移液器管空气吹。 </li><li>重新插入到操纵板肌腱，再处理完。 </li><li>验证取得的个人资料重建和横截面积的价值的形状。 </li></ol>第3部分：清洗要删除以前的操作过程中可能发生的污染，组织生物安全柜下冲洗。 3A）材料： <ul><li>冷盐溶液（D - PBS） </li><li>多槽操纵板</li><li>个人操作板</li><li>胶带</li><li> 2镊子</li><li> 1镊子站</li></ul> 3B）工作站： <ol><li>提前打开风扇在生物安全柜15分钟和清洁的内表面用70％乙醇。 </li><li>带内的生物安全柜中的所有文书。 </li><li>坚持到一个角落的每一个人板件的磁带，以确定近端。 </li><li>用无菌生理盐水溶液填充凹槽。 </li></ol> 3C）操作： <ol><li>介绍操纵板，包含在内阁中提取肌腱。 </li><li>使用镊子，取出其板块的把握上，每年年底的温柔的肌腱。 </li><li>沉浸和多槽操纵板的每个车厢的盐溶液中轻轻搅动，肌腱，从最近开始，出发到最远的凹槽。 </li><li>最后，淹没在一个单独的操纵板肌腱。 </li></ol>第4部分：载入到生物反应器室为了避免进一步污染，以下操作都是在生物安全柜中进行。 4A）材料： <ul><li>生物反应器室4 </li><li>无菌锚</li><li> 1无菌培养皿</li><li>冷战细胞培养培养基（DMEM） </li><li>瞬干胶</li><li> 2镊子</li><li>镊子立场</li><li> 0.5 -10μL微量移液器</li><li> 1标尺 <img src="/files/ftp_upload/2176/2176fig5.jpg" alt="图5" /> 生物反应器腔图5。 </li></ul> 4B）工作站： <ol><li>由于在生物安全柜，也实现了上一步，删除不必要的工具会给你更多的工作空间。 </li><li>使机柜内的所有其他文书。 </li></ol> 4C）操作： <ol><li>操纵盘集中在肌腱下肢和伤口组织其阀芯阀芯周围放置在一个锚。 </li><li>卷锚，并保持宽松的肢体，同时确保组织保持锚。 ¾转动后停止。 </li><li>进行相同与另一端的步骤。 </li><li>翻转同样两个锚，直到有6厘米之间的中心 1 。 </li><li>松开肌腱沉浸在解决方案，并允许挂靠部分简要干，以提高其机械性能2-3。 </li><li>倾入培养皿氰基丙烯酸酯，并绘制成的微量移液器2.5μL。 </li><li>在盐溶液中滴加任何胶水适用无伤口组织的氰基丙烯酸酯的下降。重复，直到2滴已应用到每个锚。 </li><li>随着肌腱仍然完全浸入，等待5分钟胶水完全干燥。 </li><li>在这段时间里，装满盐水的腔室。 </li><li>所有组织补充水分与盐溶液，以防止损坏。 </li><li>通过操纵只锚，转移到生物反应器室的肌腱和关闭，以防止在传输过程中的泄漏和污染。 </li></ol>第5部分：代表性的成果： 该协议的结果显示，当正确执行，我们的组织严密的隔离和编制程序，使人们有可能保持组织的不育性，可行性和完整性。 首先，使用简单，重复提取方法，我们能够提取不破坏的胶原蛋白网络，因为它可以由H＆E染色后提取意识到污染的部分微观分析观察的尾部筋。 <img src="/files/ftp_upload/2176/2176fig6.jpg" alt="图6" /> 图6。提取后肌腱的胶原蛋白网络（5μm的纵切面与H＆E染色） 。 然后，我们培养长达10天，并进行无菌测试的筋。每一天，我们镀使用琼脂培养液，孵育24小时。由于无细菌生长观察，我们得出的结论，我们的操作不会造成污染。 随着文件重建算法和光纤微米，我们能够估计在一个错误7 2％的保证金的横截面积。 <img src="/files/ftp_upload/2176/2176fig7.jpg" alt="图7" /> 图7。个人资料重建的RTT。 最后，我们评估使用后冲洗的步骤，并经过12天的文化时期的哺乳动物细胞的活/死可行性/细胞毒性试剂盒的可行性。由于绿色荧光活细胞大部分是很明显的的，我们可以证实，我们的隔离程序，成功地保持活组织。同样的测试，安装好后，进入生物反应器室的肌腱进行了两个小时。我们核实，脱水和胶锚没有在两个锚之间的组织传播。 <img src="/files/ftp_upload/2176/2176fig8.jpg" alt="图8" /> 图8。组织在锚可行性（绿色=活细胞，红色=死细胞）

<ol>
	<li>Bennett, M. B., Ker, R., Dimery, N., Alexander, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+various+mammalian+tendons.">Mechanical properties of various mammalian tendons.</a> J. Zool. Lond. A209, 537-548 (1986).</li><li>Dupuis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> D&#233;shydratation de matrices collag&#233;niques reconstruites in vitro: effets sur les propri&#233;t&#233;s m&#233;caniques et histologiques [dissertation]. , 114-114 (1981).</li><li>Haut, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+influence+of+strain+rate+on+the+tensile+failure+of+rat+tail+tendon.">Age-dependent influence of strain rate on the tensile failure of rat tail tendon.</a> Journal of Biomechanical Engineering. 105, 296-299 (1983).</li><li>Hupp&#233;, N., Lessard, J. -. L., Langelier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bioreactor+Design+for+the+Mechanobiological+Study+of+Soft+Tissue+Damage+and+Repair+in+Conditions+that+Provide+the+Best+Approximation+of+Actual+Use.">A Bioreactor Design for the Mechanobiological Study of Soft Tissue Damage and Repair in Conditions that Provide the Best Approximation of Actual Use.</a> , (1985).</li><li>Langelier, E., Dupuis, D., Guillot, M., Goulet, F. &. a. m. p. ;. a. m. p., Rancourt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+profiles+and+volume+reconstructions+of+soft+tissues+using+laser+beam+measurements.">Cross-sectional profiles and volume reconstructions of soft tissues using laser beam measurements.</a> J Biomech Eng. 126, 796-802 (2004).</li><li>Lee, T. Q., Woo, S. L. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+determining+cross-sectional+shape+and+area+of+soft+tissues.">A new method for determining cross-sectional shape and area of soft tissues.</a> J Biomech Eng. 110, 110-114 (1988).</li><li>Parent, G., Cyr, M., Cousineau-Pelletier, P., Desbiens-Blais, F. &. a. m. p. ;. a. m. p., Langelier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Techniques+for+the+Evaluation+of+Cross-Sectional+Area+in+Rat+Tail+Tendons+Generate+Significant+Errors.">Current Techniques for the Evaluation of Cross-Sectional Area in Rat Tail Tendons Generate Significant Errors.</a> , (1985).</li></ol>

通过应用这些实验程序，我们可以进行这样的组织在体外研究了多种。例如，组织变性的研究进行了为期10天的RTT的刺激下的应用程序。每一天，我们评估组织的力学性能，在非破坏性应力松弛试验。最后，我们能够观察到的RTT的应力变化，从而分析力学性能的进展。 <img src="/files/ftp_upload/2176/2176fig9.jpg" alt="图9" /> 图9。内河码头的应力变化评估槽松弛?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>D-PBS </td>
<td>Reagent</td>
<td>Wisent inc.</td>
<td>311-410-CL</td>
<td>Saline solution</td>
</tr>
<tr>
<td>Glucose </td>
<td>Reagent</td>
<td>Wisent inc. </td>
<td>609-037-EL </td>
<td>Saline solution
 1g/L</td>
</tr>
<tr>
<td>Antibiotics-antimycotics </td>
<td>Reagent</td>
<td>Invitrogen Corp.</td>
<td>15240-062 </td>
<td>Saline solution
 1%</td>
</tr>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Invitrogen Corp.</td>
<td>12800-017</td>
<td>Culture solution</td>
</tr>
<tr>
<td>Sodium Bicarbonate</td>
<td>Reagent</td>
<td>Wisent inc.</td>
<td>600-105-CG</td>
<td>Culture solution 3.7g/L</td>
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<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Wisent inc.</td>
<td>090150</td>
<td>Culture solution 10%</td>
</tr>
<tr>
<td>Antibiotics-antimycotics</td>
<td>Reagent</td>
<td>Invitrogen Corp.</td>
<td>15240-062</td>
<td>Culture solution 1%</td>
</tr>
<tr>
<td>Optic Micrometer</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Manipulation plate</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bioreactor chamber</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
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preparation of rat tail tendons for biomechanical and mechanobiological studies

鼠尾腱（RTT的）是一种常见的生物模型，用于在体外研究实验在肌腱的生理和肌腱领域。与这些组织的工作是具有挑战性的，因为他们是非常脆弱的，到现在为止，没有严格详细的协议，其隔离。 面对这些挑战，我们已经制定的方法和手段，以促进操纵RTT的和控制组织的可行性，不育和完整性。本文介绍了用于准备RTT的生物力学和mechanobiological研究的实验程序。我们的工作分为四个主要步骤：提取，截面积的测量，冲洗，并加载到生物反应器室。 在每一步中，所有程序，材料和操作详细介绍，使他们可以很容易地复制。此外，制定了具体的工具是：一个操纵板用于隔离RTT的，光纤微米的位置在截面积测量和锚固系统的组织到一个生物反应器的RTT的重视。 最后，我们描述了多个测试后获得的结果，来验证我们的方法。的可行性，不育性和完整性的评估表明，我们的程序十分脆弱的组织，如鼠尾肌腱操作严格。

Watch this Scientific Journal Video about Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies at JoVE.com

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

本文介绍了用于制备生物力学和mechanobiological研究大鼠尾腱的实验程序。表现出几个特点编制的主要步骤，开始提取，截面积的测量，清洗和加载到生物反应器室。

制备鼠尾肌腱的生物力学和Mechanobiological研究

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

preparation-rat-tail-tendons-for-biomechanical-mechanobiological

Research

JoVE Journal

Biology

16.8K 次观看.  Université de Sherbrooke. 本文介绍了用于制备生物力学和mechanobiological研究大鼠尾腱的实验程序。表现出几个特点编制的主要步骤，开始提取，截面积的测量，清洗和加载到生物反应器室。

视频: Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

专访：蛋白质折叠与神经退行性疾病的研究

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

科研

生物学

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

制备大鼠脑组织神经元和神经胶质发展研究总结文化

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

大鼠晶状体上皮细胞外植体的制备及文化学习终末分化

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

高度耦合的大鼠心肌线粒体的制备

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

大鼠血清适用于哺乳动物全胚胎培养的制备

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

结合表观遗传修饰和生物力学线索的两步策略，以产生哺乳动物多能细胞

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...