Name: antibody staining in c. elegans using &quot;freeze-cracking&quot;
Uploaded: 2013-10-14T21:15:00
Description: Watch this Scientific Journal Video about Antibody Staining in C. Elegans Using "Freeze-Cracking" at JoVE.com

经费是由美国国家科学基金会CCLI＃0633618和俄亥俄州大学提供。某些菌株是由线虫遗传学中心提供。研究生Reetobrata八宿出现在视频中。

注：多协议的步骤都带有用于设置和准备根据实验的具体情况选择。对于这些情况，其他的步骤都为A，B，C 等有替代措施的协议在图1中概述。 1。聚赖氨酸包被的载玻片的制备三种不同类型的幻灯片可以被使用，这取决于制备的容易性，相对的粘合性，以及抗体的非特异性结合之间的期望折衷。的滑动制剂替代细节在下面的步骤1A-1C所?...

<ol>
	<li>Harlow, E., Lane, 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> Antibodies: A Laboratory Manual. , (1988).</li><li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Transformation.">DNA Transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Boulin, T., Etchberger, J. F., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporter+gene+fusions.">Reporter gene fusions.</a> WormBook. , (2006).</li><li>Duerr, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> WormBook. , (2006).</li><li>Finney, M., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unc-86+gene+product+couples+cell+lineage+and+cell+identity+in+C.+elegans.">The unc-86 gene product couples cell lineage and cell identity in C. elegans.</a> Cell. 63, 895-905 (1990).</li><li>Garriga, G., Desai, C., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+control+the+direction+of+outgrowth,+branching+and+fasciculation+of+the+HSN+axons+of+Caenorhabditis+elegans.">Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans.</a> Development. 117, 1071-1087 (1993).</li><li>Miller, D. M., Shakes, D. C., Epstein, H. F., Shakes, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy.">Immunofluorescence microscopy.</a> Caenorhabditis elegans: Modern Biological Analysis of an Organism. , 365-394 (1995).</li><li>Crittenden, S. L., Troemel, E. R., Evans, T. C., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLP-1+is+localized+to+the+mitotic+region+of+the+C.+elegans+germ+line.">GLP-1 is localized to the mitotic region of the C. elegans germ line.</a> Development. 120, 2901-2911 (1994).</li><li>Strome, S., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+visualization+of+germ-line-specific+cytoplasmic+granules+in+embryos,+larvae,+and+adults+of+Caenorhabditis+elegans.">Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 79, 1558-1562 (1982).</li><li>Albertson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+first+cleavage+spindle+in+nematode+embryos.">Formation of the first cleavage spindle in nematode embryos.</a> Dev. Biol. 101, 61-72 (1984).</li><li>Harlow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using Antibodies: A Laboratory Manual. , (1999).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+Genetics.">Reverse Genetics.</a> WormBook. , (2006).</li></ol>

冷冻裂化协议的几种方法进行抗体染色法在C 1 线虫，它提供了一个相对简单的方法来染色蠕虫，但需要特定的试剂和练习以获得最佳效果。关键步骤（ 图1所示 ）包括：1）玻片制备（见步骤1和线虫2）手动压缩（见步骤2和3）快速固定（参见步骤3）。首先，最大限度地线虫的幻灯片粘连，最好准备，而不是依靠商业准备的幻灯片幻灯片，高分子量（长聚合物），聚赖氨?...

来确定蛋白质的细胞和亚细胞定位，科学家们已经传统上标记的组织中选定的特异性识别特定蛋白1抗体。在一些模式生物（如C）线虫 ，抗体染色常常被替换为分子遗传学技术，它更迅速地取得成果。在这些转化的生物体与构建体包含启动子和感兴趣的和绿色荧光蛋白的基因的编码区之间的基因融合物。然而，分子技术受到了一些文物，其中包括问题知道真正的启动子和高拷贝数（ 线虫的常用技术）2,3的变化构建体的表达。因此，染色的抗体仍然是许多科学家研究体内蛋白质功能的目标。 染色组织中的抗体可以是困难的，因为一个tibodies可能仅识别在特定的构象1的抗原。例如，抗体可能只承认变性或完整抗原固定在一个特定的方式，可能无法识别抗原原位 。抗体染色的线虫的问题是由线虫的角质层形成一个相对不透水的屏障，阻断抗体的获得组织中的事实而加剧。 有用于抗体染色几种不同的方法在C。线虫 （在我们以前的工作检讨4）。染色完好“蠕虫”的方法被开发出来，冻结和解冻的相对硬的固定液（甲醛或戊二醛）的冻融帮助破解角质层允许的固定液5快速渗透。固定后，角质层中透化，以允许抗体的渗透;方法包括用还原剂，胶原酶，或两者5-7治疗。这些Treatments保存形态，但往往降低或破坏抗体识别。替代方法包括解剖8，允许抗体渗透。 “冷冻裂化”是获得对半完整蠕虫的内部，以允许抗体染色9-10的方法之一。冷冻裂解可以用各种定影条件下进行，同时避免胶原酶和还原处理。线虫放置2粘合剂片之间，冷冻，然后将载玻片分离，留下大部分线虫的底部滑动和许多在顶部滑动的角质层。底部的滑动可以放置在固定剂并用粘接线虫整个滑动是通过抗体染色程序转移。该方法确实存在两大困难。首先，它是难以适用的必要的压力分裂线虫，而不会严重变形他们正确的金额。其次，许多蠕虫将不发勾选要么幻灯片，将丢失的固定液或冲洗。然而，用适当的幻灯片和实践，该方法将迅速得到线虫与合理的形态，可与各种各样的固定剂和抗体的使用。 该方法可能会稍微改变，这取决于实验者的目标。如果单线虫的染色是理想的（ 例如 ，以确定单一的转化体是否已改变的抗体染色），然后以最大的粘着滑动（但更高的背景染色）都可以使用，如实验室制备的玻片用额外多聚赖氨酸（见下文）。对于甲醛或戊二醛固定，高附着力的幻灯片（实验室制备的聚赖氨酸玻片）应该使用，因为这些线虫的注视后坚持更差聚赖氨酸。如果野生型线虫正在用甲醇固定和/或丙酮，然后以较低的粘着滑动和较低的背景染色，应使用（商业或升aboratory准备的幻灯片）。如果多种抗体和固定剂被在实验室中使用，一个选择的幻灯片可以提前准备的时间和存储，直到需要。 访问线虫组织已经获得了后，抗体染色程序遵循标准方法（较长的孵化组织的特征，而不是细胞1,11）。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine slide (ESCO brand)</td>
			<td>Fisher</td>
			<td>12-545-78</td>
			<td>Sigma brand slides also suitable</td>
		 </tr>
		 <tr>
			<td>poly-L-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P1524</td>
			<td>IMPORTANT: Brand and high molecular weight of polylysine critical</td>
		 </tr>
		 <tr>
			<td>single frosted edge slides</td>
			<td>Fisher</td>
			<td>48312-002</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>sodium azide</td>
			<td>Sigma</td>
			<td>S2002-25G</td>
			<td>TOXIC, wear gloves and mask while weighing out powder to make 10% stock. Wear gloves when pipeting stock solution</td>
		 </tr>
		 <tr>
			<td>coplin staining jar</td>
			<td>VWR</td>
			<td>47751-792</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>5-slide mailer</td>
			<td>Electron Microscopy Science</td>
			<td>71549-01</td>
			<td>One of many different brands of small slide holder, suitable for staining</td>
		 </tr>
		 <tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>MK262159</td>
			<td>IMPORTANT: Regular formalin solutions (37% formaldehyde) are NOT suitable. Either 1) Dissolve crysatlaline paraformaldehyde in phosphate buffer and store in small aliquots frozen. Or 2) buy electron microscopy grade paraformaldehyde in aliquots. TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Glutaraldehyde solution, 25% in water</td>
			<td>Sigma</td>
			<td>G 5882</td>
			<td>IMPORTANT: purchase electron microscopy grade glutaraldehyde in ampules; TOXIC -wear gloves; dispose of excess down sink with water.</td>
		 </tr>
		 <tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>EM-9410</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>ICN820451</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Donkey Serum, lyophilized</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-121</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>Cy3 Donkey anti-Mouse IgG multi-labeling</td>
			<td>Jackson Immunoresearch</td>
			<td>715-165-150</td>
			<td>This company is recommended for high quality multi-label (affinity depleted) antibodies. Select appropriate dye and antigen (animal used to raise primary antibody). </td>
		 </tr>
		 <tr>
			<td>n-propyl gallate</td>
			<td>Fisher</td>
			<td>ICN10274750</td>
			<td>Other brands are suitable</td>
		 </tr>
		 <tr>
			<td>DAPI, 4',6'-diamidino-2-phenylindole dihydrochloride</td>
			<td>Sigma</td>
			<td>D 9542</td>
			<td>TOXIC. Wear gloves and dispense into small aliquots and feeze to minimize exposure</td>
		 </tr>
		 <tr>
			<td>Whatman #1 15 cm diameter filters</td>
			<td>Fisher</td>
			<td>09805G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Coverslip #1 1/2 rectangle 24 x 60 mm</td>
			<td>VWR</td>
			<td>48393-252</td>
			<td>#1 1/2 thickness is optically best</td>
		 </tr>
		 <tr>
			<td>Microscope slide folder</td>
			<td>VWR</td>
			<td>48429-092</td>
			<td>Other brands are suitable</td>
		 </tr>
		</tbody>
 </table>

antibody staining in c. elegans using &quot;freeze-cracking&quot;

染色C。线虫与抗体的相对不透水的角质层必须通过化学或机械方法来绕过。 “冷冻破解”是指用于两个贴壁幻灯片之间压缩线虫，冻结他们，拉幻灯片除了身体拉角质层从线虫的方法。冷冻裂解提供了一种简单而快速的方式来访问该组织未经化学处理，并且可以与各种固定剂的使用。然而，它导致许多样品的损失和所需要的压缩机械地扭曲了样品。需要实践最大化具有良好的形态样品的回收率。冷冻裂解可以为特定的固定条件下，样品的回收率，或低非特异性染色进行优化，但不是对所有参数在一次。对于需要非常坚硬的固定条件和容忍需要化学通透角质层的化学处理，treatmen抗体完整的线虫在溶液中t可以是优选的。如果抗体需要一个打火机修复或如果最佳固定条件是未知的，冷冻裂解提供快速测定的抗体，可以得到特定的亚细胞和细胞定位信息对于所感兴趣的抗原的非常有用的方法。

当蠕虫被正确压缩，破裂，并轻轻固定，几乎整个蠕虫可以访问抗体染色（ 见图2）。原子核由DAPI染色所指示的位置的指示哪个蜗杆的零件都完好无损当蠕虫经受较硬的固定剂如甲醛，蠕虫的形态可能会变形（如看到的不自然的波状外形在图3A-D的肌肉）。另一个常见的​​问题是不均匀的固定或特定组织中的渗透。这样的一个例子示于图3E-H，其中的肌肉被?...

Watch this Scientific Journal Video about Antibody Staining in C. Elegans Using "Freeze-Cracking" at JoVE.com

Antibody Staining in C. Elegans Using "Freeze-Cracking"

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

“不许动开裂，”用于暴露线虫C.内组织的方法线虫抗体用于蛋白质定位，被证实。

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Research

JoVE Journal

Biology

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...