Name: a multi-hole cryovial eliminates freezing artifacts when muscle tissues are directly immersed in liquid nitrogen
Uploaded: 2017-04-06T14:00:00
Description: Watch this Scientific Journal Video about A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen at JoVE.com

31301950和31671288：这个项目是由中国国家自然科学基金委员会（NSFC）的支持。

目前的方法已经建立和验证，收获和存储超过1000个肌肉样品在我们的实验室组织学染色。所有涉及动物护理和使用程序，其次是中国农业部制定的准则。 1.设备的样品采集<ol><li>标签上的每个低温小瓶样品的身份。 注意：低温小瓶是专门设计的新鲜冷冻的组织样品的冷冻切片过程（ 图1A）。将小瓶在模具工厂（ 图1C）由聚丙烯制成。 </li><li>获得以下设备：在...

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在这里，我们描述了冷冻和储存肌肉组织进行肌肉功能的组织评估一个新的，多孔的冷冻管。在这个协议的临界修改的步骤是，在多孔冷冻小瓶的样品直接浸渍在液氮中。据我们所知，这是获取现有的冷冻方法中肌肉冷冻切片（见代表性的结果）优异的冷冻样品的最简单和最快的方式。 面对肌肉的研究人员面临的技术挑战是，冰晶最有可能使用的冷冻变硬的肌肉组织进行切?...

骨骼肌是从视营养和处理点产肉动物的最有价值的组分。在肉制品行业中，有两个特别重要的方面：肌肉生长的效率和所产生的肉的质量。由于肌肉的主要成分，肌肉纤维是直接关系到生长性能和动物1鲜肉的质量。例如，纤维的总数目（TNF）和纤维（CSAF）的截面积大多确定肌肉质量和肉品质;同时，肌纤维类型（FTC）强烈影响鲜肉的质量2。因此，肌纤维特征在动物操纵是增加核心的盈利能力和农场1的竞争力的一个非常有效的方法。 迄今为止，一些内在因素和外在因素已被确定操纵肌纤维characterist集成电路1。这种操纵可以通过动物的特定基因，的针对性地选择如在牛3 筒箭毒碱基因，绵羊4 美臀基因和RYR1和IGF2基因猪5来实现。此外，饮食控制和特定的激素治疗起到肌纤维特性6重要的作用。因此，结合遗传和营养因素的做法也许能提高瘦肉量和肉质。然而，在肌纤维的研究在肉类行业受到限制，因为肌纤维结构的阐明仍然是一个挑战。 肌纤维属性是使用组织化学方法，如肌球蛋白三磷酸腺苷酶（ATP酶）测定法鉴定。此方法依赖于酶位于的薄（6-8微米）冷冻切片的事实肌纤维可以用某些产品进行化学反应。然而，肌肉中的水含量是在猪，兔，小鼠，和人大于75％，而不管位置（ 即，背，腹，或后肢）7。在肌肉这种高水分含量导致通常遇到的问题-冷冻切片的制备过程中，如先前所描述8,9 -冷冻伪影。在大多数情况下，这几乎是不可能适当地冻结在屠宰场生产线的肌肉组织，根据我们的经验。 这里介绍的协议描述在我们的实验室用于冻结肌肉组织中高通量地冷冻切片一个简单而有效的方法。这种方法的亮点是被设计用于在液氮中快速冷冻的肌肉组织新冷冻小瓶。当前工作流可以同时促进组织冷冻和处理优异的肌肉冷冻切片，用清晰可见细胞质隔室和肌纤维的紧密并置到周围的组织。此外，该协议可以被应用到各种各样的用于组织分析选项，因为液氮不与组织混合。

<table border="0" cellpadding="0" cellspacing="0" width="508">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Cryostat Microtome</td>
			<td style="width:111px;">&#160;Leica</td>
			<td style="width:111px;">Leica CM1950</td>
			<td style="width:148px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Microscope&#160;</td>
			<td>Nikon</td>
			<td>Nikon DS-U3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">&#160;Cryogenic Vial&#160;&#160; Plastic film</td>
			<td></td>
			<td></td>
			<td>Designed by ourself</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cm-forcep</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25cm-tweezer</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Safety glass</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freezer gloves</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
	</tbody>
</table>

a multi-hole cryovial eliminates freezing artifacts when muscle tissues are directly immersed in liquid nitrogen

研究骨骼肌生理面临适当地处理标本清晰可见细胞质车厢，以获得部分技术挑战。另一个障碍是肌纤维周围组织的紧密并置。因为组织固定，石蜡包埋的过程导致肌纤维的收缩，凝固硬化是肌肉组织用于切片的最佳手段。然而，一个经常遇到的问题，冰晶的形成，因为肌肉中的水含量高的冰冻切片的制备过程中发生。这里介绍的协议首先描述了一种通过在液氮中浸渍它们适当冷冻肌肉组织的简单且有效的方法。与单独使用液氮的问题是，它会导致氮气屏障形成的组织，其充当绝缘体和抑制的组织的冷却旁边。为了避免这种“蒸汽膜”的效果，一个NE瓦特冷冻管被设计为增加周围组织表面的液体流的速度。这是通过在小瓶中的壁中的总共14个流入孔冲孔来实现。根据气泡动力学，在较小的气泡和机会较少液体流动的结果更高的速率形成气体屏障。当液氮流入通过入口孔冷冻管，围绕组织的流速足够快，以消除气体阻隔。相比于使用预先冷却的异戊烷中冷冻肌肉组织的方法，该协议是简单和更有效的，并且可以被用于在通量方式冻结肌肉。此外，这种方法最适用于那些没有获得异戊烷，这是在室温下极易燃的机构。

的冷冻管插图和普通实验室设备的冷冻切片的制备过程中冷冻肌肉示于图1。的冷冻小瓶在一个模具厂由聚丙烯制成。每个小瓶中总共具有14个流入孔：一种是在盖;另一个是在底部;和其余12形成四个平行线，每个具有在90℃到彼此的四个孔。这些入口孔可以液氮的流速加快组织旁边避免“蒸汽膜”时效果液氮接触组织。因此，很少有冰晶形成冷冻样本英寸重要?...

Watch this Scientific Journal Video about A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen at JoVE.com

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

这个协议描述的过程通过直接让他们与液氮冷冻的肌肉组织。该协议还突出一个新的离心管，可避免氮气的“毯效应”，当液氮接触样本的组织表面上。

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

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