这项研究得到了Brigham妇女医院和三月份的支持。作者要感谢Whited Lab的所有成员的支持和建议。

所有腋窝实验均按照布莱根妇女医院（BWH）机构动物护理和使用委员会进行。 1.设置灌注实验<ol><li>将成年腋窝置于装有0.1％三卡因溶液（MS222）的塑料容器中15-20分钟或直到完全麻醉。确保容器中充满足够的三氯氰胺溶液，使腋窝完全浸没。 注意:所有程序必须按照机构动物护理指南进行。在BWH时，如果脚踝测试失败，则认为腋窝完全麻醉，这意味着当脚轻轻挤压时，没有反射运动。 注意事项:尽管三奈因是特别用于水生生物的麻醉剂，但应避免与三氯氰菊酯溶液直接皮肤接触。 </li><li> 设置axolotl灌注站。 <ol><li>放置吸收垫在平坦的水平表面上，吸收面朝上。 </li><li>在聚苯乙烯泡沫框架中切割一个适当尺寸和形状的孔，使麻醉的腋窝躺在仰卧位。将框架放在吸收垫上。 注意:一些额外的纸巾可以立即放在框架下面以获得更多的吸收。 </li><li>用灌注管装入蠕动泵。将泵设置为0.7ml / min的流速，顺时针方向流动。 </li><li>在1:4混合物中用0.7倍PBS和5％葡萄糖稀释溶液。 </li><li>将10mL稀释液与200μLDiI储备溶液混合在50mL锥形管中。盖帽并通过倒置混合。用铝箔纸覆盖该管，以保护工作溶液免受光照。 注意:体积应根据腋窝尺寸按比例进行更改。这些值是一个大约15厘米的轴向长度（鼻子到尾部的长度）。一个这个大小的小数可能没有达到完全的性成熟度，所以动物的性别可能不会在这个时候确定。 </li><li>用0.7x PBS填充50mL锥形管。 注意:PBS将用于引发环和axolotl放血。 </li><li>将27号蝶形针连接到灌注管的出口端。将蝴蝶翅膀彼此折叠并放置在夹具架上。 </li><li>将灌注管的自由端放入装有0.7x PBS的50mL锥形管中，并运行灌注泵，直到整个管填充溶液。一旦整个管道充满PBS就暂停泵。 注意:确保管道始终没有气泡，因为这些将导致轴突中的空气栓塞，并防止充分灌注。 </li><li>在聚苯乙烯泡沫框架的斧形模具中放置纸巾。使用转移移液管，用三卡因溶液浸泡毛巾。 注意:在纸巾中间切一个小方块以允许在灌注过程期间排出液体。 </li><li>将麻醉的腋窝仰卧放置在聚苯乙烯泡沫框架内的纸巾上。 </li></ol></li></ol> 2.打开Axolotl胸部<ol><li>使用外科镊子沿着axolotl胸部的中心轴线夹紧皮肤，就在肩膀的下方。拉起。 </li><li>使用手术刀做一个小切口，皮肤被拉。 </li><li> 在胸部取出一个方形斑块，以露出两个软骨板。 <ol><li>去除皮肤以在胸腔上打开一个足够大的窗口，以清楚地看到心脏和大约5毫米的从心脏分支的主动脉。 </li></ol></li><li>使用镊子或封闭的剪刀小心撕开结缔组织，以避免切割任何主要的血管。 </li><li>使用镊子单独提起每个软骨板，并将其切除用外科剪刀。 </li><li>使用镊子小心地夹住心包，用手术剪刀拉起并刺穿心包;该切口应该足够深以刺穿非常薄的心包，并且应该足够大以允许去除心包。小心别切心。 </li><li>精心移除心包露出心脏和主动脉。 注意:使用转移移液管，定期用三卡因溶液冲洗胸腔和鳃，以保持区域清洁并保持腋窝麻醉。 </li></ol>灌注Axolotl <ol><li>将装载的蝴蝶针放置在聚苯乙烯泡沫框架旁边的夹具架上，使得夹具的臂能够容易地被操纵以将针头插入到轴突主动脉中。在插入过程中将针的尖端朝向动物的头部方向，并将针保持平行于主动脉，以避免穿刺通过手术正面</li><li>打开蠕动泵。 0.7x PBS应继续流经管路。 </li><li> 将针插入主动脉。 <ol><li>将主动脉弓下方的镊子滑动，稍微拉起以便于进入。 </li><li>操纵针夹组合，使得针沿着主动脉的长度延伸，向上朝向头部。使用镊子插入针头，以支持主动脉后面。 注意:针头应插入主动脉足够深，以确保灌注期间不会滑出。对于15厘米的轴突，这可能是约5毫米。确保针与主动脉完全一致，以避免完全穿刺血管。穿透穿刺可引起大量出血并降低灌注成功率。成功插入可以通过心脏心房的可见扩大来确认。 </li></ol></li><li> 用sci快速撕开一个中庭游标，让血液流失。 <ol><li>与三卡因溶液冲洗，以防止胸腔内血液积聚和血块形成。 </li></ol></li><li>用约20-30 mL PBS灌注腋窝。 成功灌注后，动物应从浅粉红色变为白色。 </li><li>暂停蠕动泵，将管道的自由端移至15 mL DiI溶液管中。重新启动泵，注意避免在管道中产生任何气泡。 </li><li>将斧头与DiI的整个工作库存完全一样。 注意:在成功的灌注中，痣样变化为DiI的明亮粉红色。这在鳃中最为显着。 </li><li>用DiI灌注完成后暂停泵，将管子的自由端放入4％的多聚甲醛（PFA）溶液中以固定组织。重新启动泵并灌注至少10 mL的PFA。 注意:PFA有毒，应处理和排除适当地。应穿戴手套和安全眼镜，并应在通风橱内进行解决。用PFA灌注腋窝与固定组织导致动物死亡。 </li></ol> 4.结束灌注和可视化准备<ol><li>停止蠕动泵，并从腋窝主动脉取出针头。 </li><li>将腋窝放在塑料板上。 注意:使用一个大的培养皿的一半效果很好，并允许将少量的三聚氰胺或PBS倾倒在腋窝上，以保持皮肤湿润并提高可视化质量。 </li><li>将所有使用的材料处理在适当的废物箱中。使用70％乙醇清洁外科手术工具，使用动物之间的玻璃珠灭菌器消毒，并按照程序通过高压灭菌灭菌。用PBS溶液冲洗管道，然后排干，充分干燥，并存放以供进一步使用。 </li></ol> Perfused Axolotl可视化<oL&gt; <li>将腋窝放在荧光共焦显微镜下。 </li><li>关闭灯泡，因为DiI染色的血管的可见性受到光的阻挡。 </li><li>使用共聚焦显微镜使用绿色荧光发射过滤器立方体（ 例如 ET-CY3）可视化轴突的脉管系统。使用波长545 nm的激发光。 注意:要获得高质量的图像，可以使用以下参数:曝光1.1秒，增益1倍，饱和度1.0，放大倍数2倍。 </li></ol>

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作者没有什么可以披露的。

可以通过用亲脂性碳菁染料DiI灌注成功地实现腋窝的脉管系统的可视化。在本研究中，我们用蠕动泵描述了用DiI灌注腋窝的新方案。我们还使用荧光共焦显微镜显示了axolotl脉管系统的随后的可视化。该方案是Li 等人所述的啮齿动物DiI灌注方案的改编。 7 ，然而啮齿动物和腋窝之间的主要差异需要修改协议以适应axolotl模型。 本研究讨论了一种Dio灌注方法，以便成功地观察脉管系统。蝾螈和啮齿动物之间的解剖学和生理学差异在灌注的主要方面需要改变，包括针插入位置，灌注方法和所用的试剂。为了ach即成功灌注，我们限制了对腋窝脉管系统造成的伤害。在打开胸腔时，要注意充分暴露心脏和主动脉，同时避免对主要血管造成任何损伤或撕裂。限制手术剪刀的使用可防止主要血管的意外夹紧，而小切口可以控制心脏和主动脉的暴露。当DiI针穿过主动脉而不是直接插入心脏的腔室时，灌注成功率也增加。与小鼠不同的是，axolotl有一个三腔心脏，只含有一个比鼠标显着更小肌肉的心室。由于这些差异，针插入的位置必须移动到更稳定的主动脉。主动脉被确定为用于插入灌注针的最佳位置，因为其足够大以用于27G针刺穿并且运动受限。运动最小以避免灌注针的意外移除或滑动或主动脉穿刺穿刺。使用心室作为插入点的心脏灌注被证明具有比具有主动脉插入点的成功率低得多的成功率。脉管系统的错误穿刺常常导致栓塞的形成或阻止灌注，导致血管标记成功率很低。灌注过程中使用夹具支撑蝶针，我们减少了运动，从而提高了灌注成功率。另外，由于腋窝组织的细腻，当与小鼠相比时，与以前使用的手动灌注相反，需要蠕动灌注泵。这种泵的使用允许对腋窝灌注的免提方法以最小化薄组织的错误穿刺。由于许多其他原因，包括穿透和穿透点，灌注不成功ure，凝血和栓塞。在将针插入主动脉并通过后壁产生第二次穿刺的情况下，DiI溶液将直接流入胸腔而不是通过全身循环。此外，一旦血液离开脉管系统，其迅速形成可能阻碍灌注的血块。脉管系统也可能形成栓塞和气泡，导致阻塞成功灌注的栓塞。最后，该方案结合了调整以适应腋窝渗透压的试剂，其显着不同于哺乳动物。该方案的适应性以及适合axolotl模型的重大变化将有助于理解再生期间组织再血管化过程。 DiI，粉红色，将灌注动物，并给它一个明亮的粉红色调。成功灌注的腋窝变得明亮的粉红色到肉眼，高度血管化区域出现更强烈的染色。使用绿色滤光片的荧光共焦显微镜观察的灌注动物可以在红橙色发射光谱中显现。脉管系统最好在更薄的组织中可视化，以最小化非血管组织的意外DiI染色。在DiI灌注后立即用4％多聚甲醛（PFA）灌注组织以固定组织。 DiI灌注是axolotl的终点实验。在手术过程中，所有动物的血液都被有效排出并用0.7x PBS替代，然后立即用DiI溶液，最后4％的PFA。这样会破坏斧头从事气体交换的重要行为的能力，并且失去了对其身体组织进行充氧的能力。由于这种终点性质，每次灌注仅捕获血管生长的单个时间点，并且动物在以后不能进一步灌注。由于这个时间限制必须使用多种动物来描述血管发育的时间过程。 该DiI协议以及用于改进DiI协议的修改可用于成功地标记和可视化axolotl的脉管系统。由于axolotl是研究再生的必需模型生物，成功的灌注开放了在再生过程中询问血管生成过程的机会。 axolotl是一种用于研究再生的模型生物，因为它是一种新生物，因此在整个成年期间保留了显着的再生能力。然而，再生组织的血运重建过程尚不清楚，因此DiI灌注对腋窝系统的适应提供了理解哺乳动物模型不可用的再生的机会。使用DiI灌注axolotl是研究revas的一种新型技术因此，该方案可以进一步用于了解疾病发育过程中的器官发生过程以及在再生过程中作为重要的工具。

脉管系统的可视化在理解许多物种生物体的结构和功能方面起着至关重要的作用。从达芬奇到十六世纪开始，研究了流通模式和图形表达方式1 。使用蜡和橡胶模具，灌注组织以产生脉管系统的三维模型，其允许研究器官发生和发病机制1,2 。树脂和蜡被染色，如印度墨水或胭脂红，以使其易于观察1，2 。然而，这些技术引起许多问题，因为它们的高粘度阻止了感兴趣组织的完全灌注1 。随着现场变得越来越复杂，使用共焦和电子显微镜发挥作用，移动灌注技术<html>远离铸模和脉管系统的液体灌注，其中一些允许血管的灌注和成像，而不会破坏初始组织3 。 DiI，荧光碳菁染料，是一种这样的污渍，允许动物的灌注而不损伤血管组织。 碳花青染料是亲油性染料，其在接触时并入细胞膜。这些染料允许血管内皮细胞容易和即时的染色，然后可以在荧光共焦显微镜下观察。 DiI通过细胞脂质膜中的侧向扩散移动，如神经元4的标记和追踪所示 。在化学上，DiI的两个烷基链赋予染料对细胞膜的高亲和力，而来自荧光染料的两个共轭环，当由绿色荧光灯过滤器激发时负责发出红色波长&gt; 4。 DiI已被广泛使用，包括成功标记质膜，并在神经元5,6 中进行顺行和逆行标记。 DiI先前已被用于灌注协议，同时可视化小鼠的脉管系统7 。 鸵鸟（ Ambosoma mexicanum ）是蝾螈，仅在墨西哥墨西哥城附近的咸水湖泊生活。这些动物已经成为理解再生过程的重要模型，因为它们可以再生全肢，尾巴（包括神经线），心脏和其他内脏的部分，以及部分眼睛作为成年人8,9 。此外，随着近来在斧头应用遗传工具，现在有可能对分子和驱动这些过程的细胞进行前所未有的洞察。成功的再培训整个肢体的配对需要广泛的血运重建过程，其可以在再生中起重要作用，而不仅仅是提供氧气和营养物质的血管的传统功能。了解组织再生背景下的血运重建势在必行。以前使用印度墨水可视化Axolotl血管，而结果令人感兴趣，这一过程在接下来的几十年中没有被重新审视。我们力求适应开发用于哺乳动物的DiI灌注协议，以允许完整的灌注和可视化的轴突脉管系统7 。该协议描述了用DiI染色技术成功灌注并随后使腋窝循环显现的步骤。该程序将允许在内稳态组织以及再生组织中专利血管的精确可视化，并提供一种新颖的视觉化方法n和腋窝血运重建过程的分析。

<table border="0" cellpadding="0" cellspacing="0" width="1583">
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			<td height="24" style="height:24px;width:405px;">Peristaltic Pump&#160;</td>
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			<td height="42" style="height:42px;width:405px;">Perfusion tubing</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">27g butterfly needle</td>
			<td style="width:195px;">EXELint Medical Products</td>
			<td>26709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">NaCl</td>
			<td style="width:195px;">AmericanBio</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">KCl</td>
			<td style="width:195px;">AmericanBio</td>
			<td>7747-40-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4&#160;</td>
			<td style="width:195px;">AmericanBio</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td style="width:195px;">AmericanBio</td>
			<td>10049-21-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Distilled water</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td style="width:195px;">AmericanBio</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:195px;">ThermoFischer</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>468495</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ethanol (100% vol/vol)</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Surgical foreceps&#160;</td>
			<td style="width:195px;">Medline</td>
			<td>MDG0748741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Polystyrene foam frame</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
			<td>any polystyrene foam square with an axolotl-shaped&#160; cut out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Surgical scissors</td>
			<td style="width:195px;">Medline</td>
			<td>DYND04025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Scalpel&#160;</td>
			<td style="width:195px;">Medline</td>
			<td>MDS15210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Absorbent underpad</td>
			<td style="width:195px;">Avacare Medical</td>
			<td>PKUFSx</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Paper towels</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Standard disposable transfer pipette</td>
			<td style="width:195px;">Fisherbrand</td>
			<td>50216954</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Clamp stand</td>
			<td style="width:195px;">Adafruit</td>
			<td style="width:152px;">291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>E10521</td>
			<td>Tricaine powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Adult axolotl</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">MgSO4</td>
			<td>AmericanBio</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">CaCl2</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">NaHCO3</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Plastic tanks</td>
			<td style="width:195px;"></td>
			<td style="width:152px;"></td>
			<td>Varying size appropriate for the axolotl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Paraformaldehyde</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Axolotl</td>
			<td style="width:195px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Leica Microscope</td>
			<td style="width:195px;">Leica</td>
			<td style="width:152px;">M165 FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">ET-CY3 Fluorescent Filter</td>
			<td style="width:195px;">Leica</td>
			<td>M205FA/M165FC</td>
			<td></td>
		</tr>
	</tbody>
</table>

dii perfusion as a method for vascular visualization in ambystoma mexicanum

灌注技术已经被使用了几个世纪来可视化的组织的循环。 Axolotl（Ambientoma mexicanum）是一种蝾螈，已经成为再生研究的基本模型。在这些动物的再生背景下，如何发生血运重建知之甚少。这里我们报告了一种简单的方法，可以通过灌注1,1&#39;-二十八烷-3,3,3&#39;，3&#39;-四甲基吲哚羰花青高氯酸盐（DiI）来观察轴突血管的脉管系统。 DiI是一种亲脂性碳菁染料，可瞬间插入内皮细胞的质膜。使用蠕动泵进行灌注，使得DiI通过主动脉进入循环。在灌注过程中，染料流经腋窝血管，并与血管内皮细胞的脂质双层接触。灌注程序大约需要一个小时，一个8英寸的腋窝。灌注后立即wi可以用共聚焦荧光显微镜观察腋窝。当用绿色荧光过滤器激发时，DiI发出红橙色范围内的光。该DiI灌注程序可用于显示腋窝的血管结构或显示再生组织中血运重建的模式。

<html>通过DiI染色，可以容易地观察到axolotl的脉管系统。在荧光共焦显微镜下立即可见用亲脂性染料灌注的动物血管。 图 1.1-1.5是灌注方案的示意图。灌满明亮的粉红色染料后，成功灌注的腋窝会出现粉红色。在共焦显微镜上使用绿色荧光滤光片会出现血管网络的红色发射。灌注成功时，所有身体组织都会发生DiI染色，包括尾巴，四肢，鳃和眼睛（ 图2A ， 图2B ， 图2C ， 图2D ）。不成功的灌注导致缺乏红色染色的脉管系统或血管斑块染色。 ntent"fo:keep-together.within-page ="1"&gt; <img alt="图1" class="xfigimg" src="/files/ftp_upload/55740/55740fig1.jpg" /> 图1:灌注方案示意图。成功灌注亲脂性染料DiI的Axolotls在成像时显示脉管系统的全部染色。灌注实验前全仰卧斧2:打开腋窝的胸部。 2:腋窝开胸腔。 3:将27 G蝴蝶针插入腋窝主动脉。 4:管道应首先含有0.7x PBS，然后是DiI工作溶液，最后含有4％PFA。 5:完全灌注的腋窝出现粉红色。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55740/55740fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55740/55740fig2.jpg" /> 图2:完全灌注Axolo的图像TL。使用DiI染色成功灌注后，使用荧光共焦显微镜拍摄腋窝脉管系统的图像。 2A:尾。 2B:脚。 2C:鳃。 2D:眼睛。使用具有绿色荧光发射过滤器立方体的共焦显微镜进行成像。图像A，B，C和D的放大倍数分别为1.74X，2.16X，1.18X和5.69X。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55740/55740fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum at JoVE.com

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

使用亲脂性1,1&#39;-二十八烷-3,3,3&#39;，3&#39;-四甲基吲哚羰花青高碘酸盐（DiI）染色技术， 墨西哥眼囊炎可以进行血管灌注，以便容易地观察脉管系统。

DiI灌注作为血管可视化的方法<em&gt;墨西哥恶性肿瘤</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

dii-perfusion-as-method-for-vascular-visualization-ambystoma

Nanyang Technological University

Research

JoVE Journal

Biology

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

9.8K 次观看.  Harvard Medical School and Brigham and Women's Hospital. 使用亲脂性1,1'-二十八烷-3,3,3'，3'-四甲基吲哚羰花青高碘酸盐（DiI）染色技术， 墨西哥眼囊炎可以进行血管灌注，以便容易地观察脉管系统。 

视频: DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

一个影像的简单方法拟南芥叶浸润成像介质中使用全氟萘烷

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.