Name: a multi-hole cryovial eliminates freezing artifacts when muscle tissues are directly immersed in liquid nitrogen
Uploaded: 2017-04-06T14:00:00.000+00:00
Duration: 6 min 42 s
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:在合适的液氮罐液氮，安全眼镜或面罩，冰柜手套，10厘米钳子，25厘米镊子，聚苯乙烯泡沫塑料冷却器（内部尺寸:20厘米长×12厘米宽X13.5厘米高;外部尺寸:24厘米长×16厘米宽×15厘米高），塑料薄膜（2.5厘米长×0.8厘米宽×0.1毫米厚），手术刀和A4 PVC结合盖板（21厘米×29.5厘米）（ 图1B）。 </li></ol> 2.肌肉样品的制备<ol><li>填充提前样品收集的液态氮5分钟的泡沫塑料冷却器。 注:液氮的大柱是必需的，因为液态氮的一部分沸腾远离而成为气体时新鲜样品触摸它。为了降低液态氮的转移频率来补充损失，适当尺寸的泡沫聚苯乙烯冷却器必须事先根据样本的数量获得的。 </li><li>一旦获得样品，轻轻将其放置在一张A4 PVC装订封面的并观察肌肉纤维的方向。 注:在这里，我们用从第一背最长肌多丽丝的横截面，以猪最后的腰椎。试样是约12厘米长，宽8厘米和5厘米高。 </li><li>用手术刀，通过试样我做两个平行切口n中的肌肉纤维的方向，在长度约3cm和0.6厘米开。修剪阴刻肌成约0.6厘米宽×0.6厘米高×1.5厘米长的矩形。 注意:测量所述肌肉纤维的截面积的可靠性主要取决于切开肌肉的纤维取向。 </li><li>使用10厘米镊子轻轻把样品在一张塑料薄膜，与肌肉平行切开的纵向轴线向膜（ 图2A）的长边。 注:该塑料薄膜具有两个功能:保持功能，有助于固定肌肉纤维的取向以及防止样品中的裂纹，由于快速冷冻粘合功能。 </li><li>使用镊子，将肌肉组织成标记的低温小瓶的下部中间，就在入口孔之间，然后紧紧盖住小瓶（ 图2B）。 </li></ol> 3.冷冻和储存肌肉样品小号<ol><li>使用25厘米镊子，迅速浸没在液氮中整个深冷小瓶在预冷却的冷却器泡沫塑料，等待直至液氮不沸腾（约10-20或多个）。 注:当液氮沸腾的冷冻管，将浮动，那么仔细沉浸在沸腾的液氮瓶10-20秒。 </li><li>转移肌肉样品至液氮储存罐或用于长期存储一个-80℃冷冻机中。 </li></ol> 4.滑动制备<ol><li>预冷低温恒温器至-20℃和-22℃之间。 </li><li>取下并丢弃旧的切片刀，向下擦拭刀架和防卷板在低温恒温器，安装在低温恒温器一个新的切片刀，而切削厚度设置为8微米。 </li><li>从液氮贮罐的深冷小瓶转移肌肉样品或-80℃冰箱到低温恒温器，在液氮或干冰上运送它。 </LI&gt; <li>等待30分钟的样品与低温恒温器的温度达到平衡。 注意:可能触摸试样的任何设备必须预冷却，因为高温可能会导致冰晶体在试样的形成。 </li><li>安装水溶性二醇和树脂（OCT）的最佳切割温度下的制剂中的试样保持器的样品。切8微米的部分。 注意:垂直调整的切割角与肌肉纤维的取向，这是因为CSAF是肌肉纤维的横截面面积。 </li><li>安装部分向室温的MicroSlide的中心。立即将滑入在-80℃冷冻机的滑动框。不要让幻灯片在室温下干燥。 </li></ol>

<ol>
	<li>Joo, S. T., Kim, G. D., Hwang, Y. H., Ryu, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+fresh+meat+quality+through+manipulation+of+muscle+fiber+characteristics.">Control of fresh meat quality through manipulation of muscle fiber characteristics.</a> Meat Sci. 95 (4), 828-836 (2013).</li><li>Lefaucheur, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second+look+into+fibre+typing--relation+to+meat+quality.">A second look into fibre typing--relation to meat quality.</a> Meat Sci. 84 (2), 257-270 (2010).</li><li>Fiems, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+Muscling+in+Cattle:+Genes,+Husbandry,+Carcasses+and+Meat.">Double Muscling in Cattle: Genes, Husbandry, Carcasses and Meat.</a> Animals (Basel). 2 (3), 472-506 (2012).</li><li>Cockett, N. E., 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=The+callipyge+mutation+and+other+genes+that+affect+muscle+hypertrophy+in+sheep.">The callipyge mutation and other genes that affect muscle hypertrophy in sheep.</a> Genet Sel Evol. 37, 65-81 (2005).</li><li>Stinckens, A., 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=The+RYR1+g.1843C&gt;T+mutation+is+associated+with+the+effect+of+the+IGF2+intron3-g.3072G&gt;A+mutation+on+muscle+hypertrophy.">The RYR1 g.1843C&gt;T mutation is associated with the effect of the IGF2 intron3-g.3072G&gt;A mutation on muscle hypertrophy.</a> Anim Genet. 38 (1), 67-71 (2007).</li><li>Brameld, J. M., Buttery, P. J., Dawson, J. M., Harper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+and+hormonal+control+of+skeletal-muscle+cell+growth+and+differentiation.">Nutritional and hormonal control of skeletal-muscle cell growth and differentiation.</a> Proc Nutr Soc. 57 (2), 207-217 (1998).</li><li>Reinoso, R. F., Telfer, B. A., Rowland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+water+content+in+rats+measured+by+desiccation.">Tissue water content in rats measured by desiccation.</a> J Pharmacol Toxicol Methods. 38 (2), 87-92 (1997).</li><li>Meng, H., 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=Tissue+triage+and+freezing+for+models+of+skeletal+muscle+disease.">Tissue triage and freezing for models of skeletal muscle disease.</a> J Vis Exp. (89), (2014).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J Vis Exp. (99), e52793 (2015).</li><li>Meijer, A. E., Vloedman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histochemical+characterization+of+the+coupling+state+of+skeletal+muscle+mitochondria.">The histochemical characterization of the coupling state of skeletal muscle mitochondria.</a> Histochemistry. 69 (3), 217-232 (1980).</li><li>Harnkarnsujarit, N., Kawai, K., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Freezing+Temperature+and+Water+Activity+on+Microstructure,+Color,+and+Protein+Conformation+of+Freeze-Dried+Bluefin+Tuna+(Thunnus+orientalis).">Effects of Freezing Temperature and Water Activity on Microstructure, Color, and Protein Conformation of Freeze-Dried Bluefin Tuna (Thunnus orientalis).</a> Food Bioprocess Technol. 8 (4), 916-925 (2015).</li><li>Dubowitz, V., Sewry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Muscle Biopsy: A Practical Approach. , 407-422 (2007).</li><li>Suvarna, S. K., Layton, C., Bancroft, J. 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> Bancroft's Theory and Practice of Histological Techniques: Expert Consult: Online and Print, 7e. , (2008).</li><li>Sulaiman, S. A., Kamarudin, N. A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bubbles+Size+Estimation+in+Liquid+Flow+Through+a+Vertical+Pipe.">Bubbles Size Estimation in Liquid Flow Through a Vertical Pipe.</a> J Appl Sci. 12 (23), 2464-2468 (2012).</li><li>Fukutomi, K., 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=A+Study+of+A+Flow+through+Small+Apertures+:+2nd+Report,+Experiments+on+The+Velocity+Field.">A Study of A Flow through Small Apertures : 2nd Report, Experiments on The Velocity Field.</a> Nihon Kikai Gakkai Ronbunshu B Hen/transactions of the Japan Society of Mechanical Engineers Part B. 53 (496), 3516-3521 (1987).</li><li>Shah, M. S., Joshi, J. B., Kalsi, A. S., Prasad, C. S. R., Shukla, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+flow+through+an+orifice+meter:+CFD+simulation.">Analysis of flow through an orifice meter: CFD simulation.</a> Chem Eng Sci. 71 (9), 300-309 (2012).</li></ol>

没有利益冲突，财务或其他方面，是作者声明。

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片的手段时形成。正如代表结果指出，有三个因素在确定病理研究，包括选择合适的冷源，最大限度地减少了样品的水分，并增加与冷源接触的总表面积的比例适当的肌肉冻结发挥显著作用。第一，冷冻速率是关键的，以避免在制备冷冻的组织的用于切片期间的损坏。当冷冻速度慢，冻结的肌肉显示出著名的"瑞士奶酪"的影响，因为大冰晶在细胞外形成。当冷冻快，但不够快，小，针状冰晶大量的单个肌肉细胞内形成。这些针状冰晶导致可检测的小的结构损坏，但它们带来了以下效果:线粒体损坏10中，肌纤维的尺寸被人为地增加11，和纤维结构的完整性被破坏11。因此，在足够冷的温度的冷源和足够的冻结速度是必不可少的，以避免在肌肉冷冻切片冰晶的形成。 理想情况下，冷源是在极低的温度下，并且被布置吨O触点所有表面。两个液氮（-190℃），异戊烷在固 - 液混合状态（-160℃）满足该条件。然而，这两个冷冻媒体有自己的缺点。液氮具有极低的比热常数。其结果是，液态氮和组织之间的接触导致该积聚旁边的组织和大大减慢的冷渗透到组织12上的蒸汽屏障。因此，会出现冰冻肌肉内显著冻结文物。异戊烷的淤浆状态是一种理想的冷冻介质不形成蒸气的障碍，但主要问题是，异戊烷必须用液氮结合以达到160℃的液体浴的温度下使用。此外，异戊烷在室温下是极其易失性和易燃液体。其次，肌含有过量水（&gt; 75％），所以任何方式将外源水分进入样品<html>会导致冻结人为效应。例如，华侨城安装中增加了额外的水到肌肉，使冰晶在那些肌肉冰冻切片更为突出与那些没有安装华侨城相比，中等。最后，肌肉组织的厚度也是重要的适当冷冻它，因为冷冻的速率部分地取决于总表面的与冷源（通常，厚度&lt;0.5厘米）接触的百分比。因此，现有唯一的方式来适当地冻结所述组织是沉浸肌肉组织的厚度小于0.5cm到预冷却的异戊烷（160℃），如先前所描述8。 该协议首先介绍如何正确地通过直接在液氮中浸渍它们冷冻肌肉组织。与单独使用液氮的问题是氮气在组织表面上形成，作为绝缘体和抑制的组织的冷却F"&gt; 13，这样的蒸汽膜只能形成时气泡的合并是如此之快，氮气可以分离从液氮组织。根据气泡动力学，更快的液体流将导致更小的气泡，因为它具有较少机会合并14。因此，我们冲孔14米的孔进入冻存管的壁，以增加围绕组织的液体流的速度，并帮助避免"蒸汽膜"的效果，当液氮接触组织。 考虑到确定实验成功的一个因素是组织周围的液体流动的速度，冷冻小瓶的三个参数是非常重要的，包括位置和孔数，入口孔的直径，并且孔之间的距离和例子。液体动力学表明，通过孔的流速通过的孔的直径来确定，并且再循环流发展立即向下孔的流（ 图5）15，16。相应地，孔和样品之间的距离大多确定高速流体流和样品之间的接触面积， 如图5。因此，在图1A中的多孔冷冻管适合于肌肉样本而为0.6厘米高，长宽1.5厘米×0.6厘米对于较大的肌肉的片段，这是值得尝试这种方法具有较大的管和更多的孔。然而，该协议不适合于从冷冻小动物，如在鼠模型肌肉样品，因为入口孔可能是更小的样品太大。 此外，有三个关键步骤必须严格遵守的协议。首先，将液氮应足以浸没整个冷冻管，并在该低温小瓶必须完全低于液体水平。其次，它是必要的冷冻的肌肉和室温容器或器具之间空隙的意外接触。例如，如果没有小心运输，新冰晶可能形成从液氮贮罐输送当冷冻样本或-80℃冰箱到低温恒温器。第三，不应该有当试样安装在保持器由于周围冷冻肌肉和OCT之间的接触面积冰晶形成的可能性高太多OCT使用。 在肉类工业，肌肉冷冻切片过程是必不可少的，因为福尔马林固定，石蜡包埋的过程引起收缩的肌肉纤维，这损害一些肌肉性状，如TNF和CSAF的评估。此外，采用肌球蛋白ATP酶试验评估FTC时，需要冷冻的肌肉。 如图3E所示，该协议可以同时满足病理分析的和在肌肉组织化学要求污渍。准备一个冷冻切片的是在临床肌肉病理实验室的例行操作。如由Meng 等人描述。 8，用于冷冻肌肉组织一个标准操作程序是在预冷却的异戊烷沉浸尸检标本。然而，异戊烷不可冻结在机构尸检标本无临床肌肉病理实验室。因此，诊断肌肉活检常常未达最佳的在许多医疗机构进行处理。因此，我们的协议可以大大改善小型医院或实验室进行的足够的质量诊断肌肉活检的能力。考虑到高效率和这个协议的简单的操作，它可能是一个有希望的替代方案，目前基于异戊烷冷冻方法，可以广泛应用到在未来组织学研究。

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><tbody><tr><td>Cryostat Microtome</td><td>&amp;nbsp;Leica</td><td>Leica CM1950</td><td></td></tr><tr><td>Digital Microscope&amp;nbsp;</td><td>Nikon</td><td>Nikon DS-U3</td><td></td></tr><tr><td>&amp;nbsp;Cryogenic Vial&amp;nbsp;&amp;nbsp; Plastic film</td><td></td><td></td><td>Designed by ourself</td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>Scalpel</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>10 cm forceps</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>25 cm tweezer</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>Safety glass</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>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的是，异戊烷，极易失性和易燃液体，在28℃的沸点，没有必要在此过程。 为了进一步突出这种方法的优势，我们冻结了背最长肌在猪中使用五个冷冻方法，然后观察其染色性质和使用苏木精/曙红（HE）染色（ 图3）组织学的信息。经常遇到的问题是冰晶的肌肉冰冻切片的制备过程中形成的。当样品在-80℃冷冻机中（ 图3A）直接放置，大冰晶形成，然后导致在肌肉冷冻切片一个公知的"瑞士奶酪"的效果。此外，当肌肉组织在液氮中（ 图3B）直接浸入也形成冰晶。即使液体氮是非常冷的液体（-190℃），它的热常数极低。当任何样品接触液体氮，蒸汽毯可旁边的组织建立和绝缘的冷渗透到组织中。另外，对于在病理学实验室冷冻标本常规方法是尚未适用于肌肉组织。一个š 如图3C中所示，形成于肌纤维的细胞质隔室中的肌肉组织中浸渍后到OCT封固剂以正确的方向，然后在异戊烷中的浆料中快速冷冻许多针状冰晶（-160℃）。总之，对肌肉组织相应的冻结率必须严格在操作满足。 当前协议实现出色的试样完整性，具有清晰可见的细胞质隔室和肌纤维的紧密并置到周围组织（ 图3D）。它允许使用肌球蛋白ATP酶试验（ 图3E）肌纤维的代谢特性的优秀的可视化。对于该测定法，肌球蛋白ATPase活性是基于这样的事实:快速收缩肌纤维（哺乳动物类型2A和2B）水解ATP比慢收缩纤维（哺乳动物1型）更快的分析。当给equiva借给倍，快收缩纤维出现光，用两个度，慢收缩纤维显示为黑色。重要的是，这种方法可以有效地在高通量环境中使用，因为它容易进行并节省了时间（ 图4）。如今，一个完善的冷冻法是浸没肌肉组织中的异戊烷不与OCT安装介质接触的浆液，如先前所描述8,9（ 图3F）。在此过程中，它需要为240毫升的异戊烷中的固-液浆料状态近似30分钟至通过冷却预在液氮中异戊烷并搅拌直至小的白色沉淀出现在底部（图4）来实现。存在由研究者遇到在试图冻结预冷的异戊烷肌肉组织3个实际困难:（1）isopantane的固 - 液浆料状态难以维持当采样时间大于1个小时。在我们过去的努力，冻结人为效应通常发生在这个过程中，当收集一次几十肌肉组织的中期和晚期阶段都做到了样本英寸（2）作为危险液体，isopantane不会在任何位置上的公共交通允许的。 （3）异戊烷是除了在实验室不可用。许多肉类科学的研究人员通常在屠宰场是远离实验室和不允许有异戊烷冻结肌肉组织。此外，诊断肌肉活检由于在医疗设施缺乏异戊烷的往往是次最佳处理。因此，我们的协议是在异戊烷是不可用的情况冰冻肌肉标本的一个重要发展。 <img alt="图1" src="/files/ftp_upload/55616/55616fig1.jpg" /> 图1:肌肉冻结的装置。 （A）中的插图在这项研究中使用的离心管。 14个入口孔被冲压在管壁: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。 （B）的冷冻装置的照片。在液氮中冷冻的肌肉的安全和有效的方法包括使用离心管，塑料薄膜，安全眼镜，冰柜手套，10厘米钳子，25厘米镊子，聚苯乙烯泡沫塑料冷却器，解剖刀，和A4 PVC装订封面的。 （C）由工程师，扬瓦·沃，在模具工厂提供的冷冻管模具的说明图。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55616/55616fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55616/55616fig2.jpg" /> 图2:肌肉样品的制备。 （A </强&gt;）将切开的肌肉组织上的塑料薄膜。 （B）所述的肌肉组织应入口孔之间在低温小瓶放置。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55616/55616fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55616/55616fig3.jpg" /> 图3:之后他们用5种方法冻猪背长肌的肌肉组织学评价的代表图像。 冰晶破坏HE染色时的肌肉使用不适当的方法冷冻，用于组织学分析，如图（AC）成像肌肉冷冻切片的可读性。当肌肉样品直接置于80℃的冰箱中（A）中或在液体中浸渍时发生相当大的冷冻伪影氮（B）。肌肉组织浸入十月后，然后直接放入异戊烷（C）的淤浆个体肌肉细胞内形成许多针状冰晶。在本研究中提出的协议可以消除冰晶的形成时的肌肉组织被简单地浸在液体氮（D）。该协议是适用于基于所述肌球蛋白ATP酶试验（E）的酶活性的冷冻肌肉分析。肌肉不会有更好的表现标准的冷冻方法;肌肉组织浸渍在异戊烷中的浆液而不与OCT安装介质（F）直接接触。所有图像是下10X物镜，除了那些与黑边（20X）。比例尺:100微米。在图3C中，箭头表示小，针状冰晶。在图3E的箭头和指示文本肌纤维的类型:类型1代表慢肌球蛋白重链的纤维，叔YPE 2A意味着中间肌球蛋白重链的纤维，并键入2B表示快肌球蛋白重链的纤维。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55616/55616fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55616/55616fig4.jpg" /> 图4:用于基于液体氮和基于异戊烷冷冻程序的工作流程的比较。在液体氮基协议，在新的离心管肌肉组织浸没在液氮中。在基于异戊烷的协议，异戊烷的浆料状态必须事先通过预冷于液氮的异戊烷的并搅拌直至小，白色沉淀出现在底部来实现。然后，肌肉组织浸入预冷的异戊烷。与基于异戊烷过程35分钟相比，如在图3F中所描述的，只需要5分钟吨<html> O的在这个协议中提出的程序冻结。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55616/55616fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55616/55616fig5.jpg" /> 图5:液氮流过锐孔。当浸渍在液氮中，大气压力迫使液体氮整个多孔冷冻管到管通过14个进入孔。孔的存在使得流加速通过它们，因此提高了速度。再循环流（蓝色）开发立即孔口（橙色）的下游。 D1表示孔口在管的壁的直径和D2表示在孔口和肌肉样品（红色）之间的距离。="_空白"&gt;点击此处查看该图的放大版本。

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 ...

a-multi-hole-cryovial-eliminates-freezing-artifacts-when-muscle

Universidad San Sebastian

Research

JoVE Journal

Biology

11.3K 次观看.  Jiangxi Agricultural University. 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。 

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

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

用于单颗粒低温电子显微镜的生物标本的手动印迹和切入式冷冻

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

科研

生物化学

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

医学

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

用于神经肌肉分析的大鼠声带的制备

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

神经科学

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

生物学

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

骨骼肌中脂质液滴含量的纤维类型和亚细胞特异性分析

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

暴跌冻结:在透射电子显微镜的超微结构和免疫组化研究工具悬架细胞

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Regulating Temperature in the Lab: Preserving Samples Using Cold

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs. This video will discuss some of the methods for keeping laboratory samples cold and will explain the correct cooling method to use for each experimental requirement.
For example, cooling agents, such as ice and dry ice, are typically used when keeping samples cold during experiments. This video discusses the physical properties of the most commonly used cooling agents, as well as safety precautions for working with them.
When it comes to keeping samples cold in between experiments, cooling equipment, including laboratory grade refrigerators and freezers can be used to preserve samples for extended period of time. Also discussed in this video are types of samples and reagents that can be stored in the commonly-available laboratory cooling equipment.
Finally, the concept of cryopreservation is introduced as a process through which tissues, cells, and biomolecules are cooled to sub-zero temperatures, thereby effectively stopping all sample-degrading biological activity. Several methods of cryopreservation are discussed that minimize or eliminate the formation of damaging ice crystals.

Preserving Samples Using Cold: Cooling Agents and Instruments, and Cryopreservation

实验室中的温度控制:冷却保存样品

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs.  This video will ...

教育

JoVE Science Education

基础生物学

常用的实验室技术

Snap Freezing of Muscle Tissue for Sectioning: A Protocol to Obtain Ultrathin Sections of Rapidly Frozen Murine Muscle Tissue

Source: Bonetto, A. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/54893" target="_blank">The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia.</a> J. Vis. Exp. (2016)
This video describes the procedure for snap freezing muscle tissue and obtaining its thin sections. The technique can help study cancer-related cachexia in colon cancer mouse models.

用于切片的肌肉组织快速冷冻:获得快速冷冻小鼠肌肉组织超薄切片的方案

Source: Bonetto, A. et al. The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia. J. Vis. Exp. (2016)
This video ...

JoVE Encyclopedia of Experiments

癌症研究

Gastrointestinal Cancer

Animal model studies

Cryopreservation of Murine Brain by Snap Freezing: A Technique to Rapidly Freeze Whole Mouse Brain Specimen for Histological Analysis

Source: Comba, A. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular">Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a> J. Vis. Exp. (2020)
This video demonstrates the cryopreservation of a mouse&#8217;s brain to isolate discrete anatomical areas or specific cell populations from the tumor tissues. This technique is critical in obtaining optimal optical resolution of brain tissue morphology for subsequent analysis.

通过快速冷冻冷冻小鼠脑冷冻:一种快速冷冻小鼠全脑标本用于组织学分析的技术

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...