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?...

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的是?...

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 de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K 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

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

冻结人类胚胎干细胞

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

科研

生物学

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

闪存冻结和Cryosectioning E12.5小鼠脑

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

冷冻，解冻，和运输包装细胞

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 &#177; 0.30 &#956;m/sec in R256H cells compared to 1.60 &#177; 0.42 &#956;m/sec in wild type cells, p &#60; 0.005).

Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.

Aip1p动力由R256H突变的肌动蛋白蚀变

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of ...

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 ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

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 ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

吸收荧光、磁性纳米微粒测定昆虫的吸液能力

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...