我们要感谢我们的顾问，博士除夕黄鼠狼。这项研究是由国家卫生赠款NS17813前夕黄鼠狼和NS059255研究所的支持曝光T。

龙虾的肚子开始前这个协议必须从动物中删除，并准备在同伴朱庇特的文章（比尔曼与托宾2009年）所述。下面列出的步骤大​​部分是根据解剖镜下进行。 我隔离的连合核<ol><li>东方的菜，所以，前年底（唇）远离您和后部（幽门）对你。 </li><li>确保胃固定，使菜就在于尽可能持平。 <ol><li>唇两侧，牵制食道。 </li><li>如果有必要，重新排列放置在总剥离的其他引脚。 </li></ol></li><li>取出的皮下组织。 <ol><li>抓斗皮下​​组织附近的gm2/gm3肌肉，这些肌肉分离和前方向轻轻拉。 </li><li>剪断任何连接，从底层的脂肪分离皮下组织。的使用增加了照顾围绕中央动脉，其中封闭的stomatogastric神经系统的重要组成部分。保留完好的约0.5厘米半径的stomatogastric节，位于之间的gm1b肌肉内的皮下组织。 </li><li>切断分离皮下组织。 </li></ol></li><li>从底层组织分离的大脑。 <ol><li>确定两个大型项目由前脑的部分双边在胃的前部连合神经节连合神经。 </li><li>让这些神经完好无损，用血管钳轻轻提起大脑和削减其他组织分开的大脑。要小心，因为你这样做，stomatogastric神经是大脑和下必须保持完整。 </li></ol></li><li>从周围的组织分离的连合核。 <ol><li>一个连合神经连合神经节之一。 </li><li>确定离子和儿子 -这些神经必须保持不变。连合节将有许多神经，辐射出来。剪下其中的大部分（ 除离子和儿子），但留下一个额外的附加 ​​举行的地方连合神经节，当你完成剥离的神经。 </li><li>重复与其他连合神经节。 </li></ol></li><li>清洁离子和儿子从外侧向内侧。离开唇神经完整无缺地锚的神经系统，胃。食管神经节位于离子小号相交。您可以清除它下面抓住伪劣心室神经拉起切底。 </li></ol>第二部分隔离Stomatogastric节（STG）和剩余的神经。 <ol start="7"><li>从周围的组织分离的STG。 <ol><li> STG坐在里面gm1b肌肉上下的gm2a，B肌肉之间运行的动脉。寻找附近的gm2a，B肌肉动脉切开结束。 </li><li>拉和前方的动脉切开年底，揭露DVN。继续向上提拉，直到您到达其余皮下组织的水平。一旦分离，动脉可修剪的水平，倒是其余皮下组织。 </li><li>切断位于肌皮瓣在主席台上的龙虾。 </li><li>抬起皮下组织以上的动脉，观察动脉内脂肪以上STG的隧​​道。把里面的动脉你的剪刀刀片和削减它上面的肌肉。检查，你可以看到DVN，你砍，以确保您安全STG以上。继续向前切割，下面的动脉。正如您上面STG切，你将能够看到的STN从STG和俯冲而下进入肌肉产生前方。继续切割前方以上STG肌肉完全独立。 </li><li>清除任何组织离了STN。 </li></ol></li><li>从周围组织中分离 AGN小号。 （即使你不打算从AGN记录，你将需要一个完整的神经部分牵制神经节细胞内录音。） <ol><li>为了更好地看到 AGN小号，除去剩余的皮下组织和一层薄薄的底层肌肉。 </li><li>就在STG后，发现一个黑暗的隧道中的脂肪和肌肉的DVN两侧。 </li><li>坚持一个隧道内的剪刀刀片，切断上述神经组织。继续横向切割，神经，孤立。 （一对夫妇毫米足以寄希望于神经节，但如果您打算从它来记录，揭露至少5毫米的 AGN。 ） </li><li>由它支配的肌肉分离，切割连接 AGN 。 </li><li>在另一侧，重复隔离其他 AGN 。 </li><li>一旦双方AGN有被解剖，切断所有连接STG胃的肌肉和组织。 </li></ol></li><li>隔离MVN号他们是一双小神经分支LVN和横向穿越下方的脂肪双边。 <ol><li>无论是用剪刀或两个钳穿过脂肪的DVN的一侧，靠近它分支到两个 LVN第在这里，你是远离MVN小号，并不会削减他们的风险。 </li><li>抬起的脂肪层，并确定下面的小 MVN 。这些神经运行内侧的gm1b肌肉水平横向后。 </li><li>拆下的MVN以上的脂肪层，使神经不变，所以现在也不会成为纠缠不清。关闭胃解剖结束，您将削减其外侧端。 </li><li>重复对方的过程中，隔离等 MVN 。 </li></ol></li><li>隔离的LVN S和后部的神经。 <ol><li>开始LVN s分支横向关闭的DVN，后续通过削减它上面的脂肪LVN。根据编制，有时整个脂肪层以上的神经被拉断。 </li><li>继续揭示，直到在PSN分支点的LVN。 </li></ol></li><li>隔离的pyn。 <ol><li>找到的pyn，它是一种神经向后行驶超过幽门。 </li><li>取下薄膜覆盖本地区。 </li><li>切割幽门的一小部分，附着的 pyn 隔离 pyn 。 </li><li>轻轻抬起幽门部分，并剪去的 pyn两侧组织，直到您到达分支连接到 PDN 。 </li></ol></li><li>隔离PDN。 <ol><li>找到PDN，它是一个向后行驶到幽门扩张肌的神经。 </li><li>幽门扩张肌肉切一小部分，连接到PDN。 </li><li>轻轻抬起幽门扩张肌节，剪去的PDN两侧的组织，直到你到达分支的 pyn重视。 </li></ol></li><li>隔离腹LVN，pyn和PDN连接的LVN。 <ol><li>在三角形的分支点 pyn 和 PDN连接到腹侧 LVN，隔离任何一小部分神经分支， 如 vpln，关腹 LVN 。这将是作为一个处理拉腹LVN。 </li><li>电梯上的处理和剪去腹LVN两侧的组织，工作前的方向，直到您到达 PSN分行关闭的地步。 </li></ol></li><li>重复步骤10 - 13，在另一侧的隔离 LVN和后的神经。 </li><li>从胃分离stomatogastric的神经系统。 <ol><li>要充分分离胃LVN，拉PSN和LVN和腹侧LVN，除为 PDN 和 pyn，削减所有的组织和神经。在另一边重复这个过程。 </li><li>剪下每个MVN外侧端。电梯外侧端，降低神经和底层的胃组织之间的任何多余的附件。 </li><li>前年底，削减任何多余的附件的连合核，每边切唇神经。 </li><li>目视扫描stomatogastric神经系统的系统来识别，然后切胃组织的任何其他附件。 </li><li>抓斗上的两个连合神经前结束，以上的连合核，剥离stomatogastric关闭胃的神经系统，将其移动到一侧的菜。当你这样做，削减任何剩余的附件，以免费的神经系统。 </li></ol></li><li>准备小Sylgard菜。 <ol><li> Sylgard小碟表面彻底擦些胃组织。这将减少对Sylgard的疏水性，使生理盐水将均匀分布在菜。 </li><li>用冷冻盐水冲洗，并填写Sylgard菜。 </li></ol></li><li>抓住两个连合神经前结束，解除神经系统的大型盘，和铺设在小碟子里，神经系统转移的准备Sylgard菜。 </li><li>引脚小碟STNS使用minuten引脚和钨丝，切更薄引脚的组合。 <ol><li>确保编制背侧检查，劣质心室神经指向距离的Sylgard。 </li><li>取出大脑和引脚连合神经。 </li><li>轻轻拉动拉在PSN S的后方和侧面，寄托他们神经系统的紧张和平面。 </li><li>删除从PDN小号幽门扩张的肌肉，和针 PDN第</li><li>取出幽门段pyn小号，和PIN pyn号</li><li>牵制任何其余孤立的神经，如唇神经，MVN s和AGN号</li></ol></li><li> Desheath的STG。 <ol><li>确保STG是平对Sylgard和横向AGN S，前STN和后DVN举行绷紧。 </li><li>排列的灯光，使可视化的轴突内的 DVN 。 </li><li>使用小针或细的钨针，使一个浅孔小，在周围的天柏，只是后的STG鞘。 </li><li>拉整个DVN针，背的轴突，神经鞘创建一个横向撕裂。 </li><li>抓住皮瓣神经鞘后的STG和向前拉删除的STG背侧鞘。 </li></ol></li></ol><table border="1"><tbody><tr><td colspan="2"> 缩写： </td></tr><tr><td> STNS </td><td> Stomatogastric神经系统</td></tr><tr><td> STG </td><td> Stomatogastric节</td></tr><tr><td> COG </td><td>连合神经节</td></tr><tr><td> STN </td><td> stomatogastric神经</td></tr><tr><td> MVN </td><td>内侧心室神经</td></tr><tr><td> 离子 </td><td>劣质食管神经</td></tr><tr><td> 儿子 </td><td>优越的食管神经</td></tr><tr><td> AGN </td><td>前胃神经</td></tr><tr><td> DVN </td><td>背心室神经</td></tr><tr><td> LVN </td><td>侧脑室神经</td></tr><tr><td> PSN </td><td>后胃神经</td></tr><tr><td> vpln </td><td>腹后外侧神经</td></tr><tr><td> pyn </td><td>幽门神经</td></tr><tr><td> PDN </td><td>幽门扩张神经</td></tr></tbody></table>

<ol>
	<li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generators+and+the+control+of+rhythmic+movements.">Central pattern generators and the control of rhythmic movements.</a> Curr. Biol. 11, R986-R996 (2001).</li><li>Nusbaum, M. P., Beenhakker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-systems+approach+to+motor+pattern+generation.">A small-systems approach to motor pattern generation.</a> Nature. 417, 343-350 (2002).</li><li>Clemens, S., Massabuau, J. C., Legeay, A., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+modulation+of+interacting+central+pattern+generators+in+lobster+stomatogastric+ganglion:+influence+of+feeding+and+partial+pressure+of+oxygen.">In vivo modulation of interacting central pattern generators in lobster stomatogastric ganglion: influence of feeding and partial pressure of oxygen.</a> J. Neurosci. 18, 2788-2799 (1998).</li><li>Bierman, H. S., Tobin, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gross+dissection+of+the+stomach+of+the+lobster,+Homarus+americanus.">Gross dissection of the stomach of the lobster, Homarus americanus.</a> J Vis Exp. 27, (2009).</li><li>Maynard, D. M., Dando, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus,+Homarus+americanus+and+Panulirus+argus+(Decapoda+crustacean).">The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus americanus and Panulirus argus (Decapoda crustacean).</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 268, 161-220 (1974).</li><li>Herrick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+the+American+lobster.">Natural history of the American lobster.</a> Bull. U.S. Bur. Fish. 29, 219-252 (1909).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs.">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Annu. Rev. Physiol. 69, 291-316 (2007).</li><li>Bucher, D., Taylor, A. L., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generating+neurons+simultaneously+express+fast+and+slow+rhythmic+activities+in+the+stomatogastric+ganglion.">Central pattern generating neurons simultaneously express fast and slow rhythmic activities in the stomatogastric ganglion.</a> J. Neurophysiol. 95, 3617-3632 (2006).</li><li>Mizrahi, A., Dickinson, P. S., Kloppenburg, P., F&#233;nelon, V., Baro, D. J., Harris-Warrick, R. M., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+maintenance+of+channel+distribution+in+a+central+pattern+generator+neuron+by+neuromodulatory+inputs+revealed+by+decentralization+in+organ+culture.">Long-term maintenance of channel distribution in a central pattern generator neuron by neuromodulatory inputs revealed by decentralization in organ culture.</a> J. Neurosci. 21, 7331-7339 (2001).</li><li>Schulz, D. J., Goaillard, J. M., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+channel+expression+in+identified+single+and+electrically+coupled+neurons+in+different+animals.">Variable channel expression in identified single and electrically coupled neurons in different animals.</a> Nat. Neurosci. 9, 356-362 (2006).</li><li>Schulz, D. J., Goaillard, J. M., Marder, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+expression+profiling+of+identified+neurons+reveals+cell-specific+constraints+on+highly+variable+levels+of+gene+expression.">Quantitative expression profiling of identified neurons reveals cell-specific constraints on highly variable levels of gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 13187-13191 (2007).</li></ol>

龙虾胃的解剖和STNS已经很好地描述以前 5,6，并且已在许多研究利用这一解剖过程中的变化。我们建议变得熟悉5-7 STNS 5,8前开始解剖胃 ​​的解剖。这种熟悉会减少意外损坏的神经和组织的风险。 为了保持健康的准备，它是一个很酷的温度保持在编制的关键。这是很容易实现交换生理盐水与4˚彗星生理盐水在冰箱中保持每30分钟一班。当沉浸在冰冷的生理盐水，隔离STNS准备是可行的多个小时。在相关Homarus钩虾的录音表现出平稳有节奏的活动从STG各地在体外 九日类似的稳健性已在 H americanus（人数OBS）。。 从连合核STG调变输入需要保存正在进行的电机模式。因此，要保持一个正常的节奏这些活动节和离子，儿子， 和 STN神经，必须保持完整。我们所描述的解剖保留了一些运动神经（含运动神经元轴突）：AGN小号的（含胃磨（GM）的轴突），MVN S（含横向胃（LG）和劣质的心脏（IC）的轴突 ） ，pyn小号（含幽门神经元（PY）轴突），PDN S（含幽门扩张器（PD）轴突）， 和 LVN S（含横向幽门（LP），内侧胃（MG），液化石油气轴突以及那些从通用汽车公司， PD和PY）。通过解剖额外的（或不同）的神经，运动神经元轴突可能被记录下来。每个运动神经元轴突的个人可单独录制，通过解剖和记录从神经支配的肌肉，这种类型的神经元项目。 本制剂可用于各种研究侧重于神经系统的功能。使用somata或轴突细胞内，夏普电极录音，或通过记录电脉冲从神经细胞外的神经元的活动可能会被记录。部分已在许多STG神经元离子通道的克隆和表达，可以从手动拉 somata 10,11测量。

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Tool</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Cut into 1-2mm pieces to use as fine pins.</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Tool</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Tool</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Tool</td>
<td>FST</td>
<td>15370-52</td>
<td>These are the top of the line. There are more economical spring scissors available from the same company.</td>
</tr>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Tool</td>
<td>Fischer</td>
<td>08-757-12</td>
<td>Fill 2-4mm with Sylgard 182.</td>
</tr>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Tool</td>
<td>FST</td>
<td>26018-17</td>
<td>Optional. Desheathing can be done with scissors and fine forceps alone.</td>
</tr>
<tr>
<td>Super fine forceps #5SF</td>
<td>Tool</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Tool</td>
<td>FST</td>
<td>10130-05</td>
<td>Optional. Desheathing can be done with scissors or with pins cut from fine tungsten wire.</td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

homarus americanus stomatogastric nervous system dissection

了解神经系统的生产活动和应对环境的目标，神经学家模型系统表现出兴趣的活动和实验方法，并且可以。 stomatogastric神经系统（STNS）（美国龙虾<i&gt; Homarus americanus</i&gt;;也知道是大西洋或缅因州龙虾）已被确立为一个模型系统，网络和网络的神经调节学习节奏。 STNS由前神经节（2连合神经节和食道神经节），其中包含的调节神经元的项目集中stomatogastric节（STG），。 STG包含约30个神经元，包括两个中心的格局产生网络，幽门及胃网络基础中的甲壳类动物的喂养行为<sup&gt; 1,2</sup&gt;。虽然有可能为研究本系统<i&gt;在体内</i<sup&gt; 3</sup，STNS继续隔离时产生的节律活动<i&gt;在体外</i&gt;。在培养皿中的STNS的物理隔离可以轻松访问，神经节细胞内的电生理记录somata外录音STNS神经。隔离STNS是由两部分组成的过程。第一部分，从肚子里的动物解剖，在随后的视频文章介绍<sup&gt; 4</sup&gt;。在这个视频文章，精细分离技术被用来隔离从胃STNS。这一过程的结果在神经电生理记录系统准备。

Watch this Scientific Journal Video about Homarus Americanus Stomatogastric Nervous System Dissection at JoVE.com

Homarus Americanus Stomatogastric Nervous System Dissection

我们描述了罚款清扫stomatogastric从胃的美国龙虾的神经系统（<i&gt; Homarus americanus</i&gt;）。

Homarus Americanus Stomatogastric神经系统解剖

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

homarus-americanus-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Homarus Americanus Stomatogastric Nervous System Dissection

11.1K 次观看.  Brandeis. 我们描述了罚款清扫stomatogastric从胃的美国龙虾的神经系统（ Homarus americanus）。

视频: Homarus Americanus Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

从中央与EAE大鼠中枢神经系统的单核细胞的分离

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of cells (approximately 25 in C. borealis) which are well characterized. The cells in the STG exhibit a broad range of outputs and are responsible for the motor actions of the stomach. The stomach contains the gastric mill which breaks down food with three internal teeth, and the pylorus which filters the food before it reaches the midgut. The STG produces two rhythmic outputs to control the gastric mill and pylorus known as central pattern generators (CPGs). Each cell in the STG can participate in one or both of these rhythms. These CPGs allow for the study of neuromodulation, homeostasis, cellular and network variability, network development, and network recovery.
The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

癌症北欧化工 Stomatogastric神经系统解剖

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

从人骨关节炎膝关节收集和RNA提取

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...