我们非常感谢黄鼠狼实验室成员。我们感谢她的支持和鼓励我们的顾问，除夕黄鼠狼，博士。这项研究是由美国国家神经疾病研究所和NS - 17813和NS - 058110中风资助支持。

1。毛夹层<ol><li>在冰上放置30分钟沉闷的动物的感觉和运动系统的螃蟹。用冰各方环绕它。处理蟹时，必须戴上手套。 </li><li>总值剥离准备铺设夹层锅，您将使用的工具。 Rongeurs，骨切割剪刀，小剪刀，有齿镊，锅铲锥形边，一个黑色的Sylgard涂层盘，和昆虫引脚是必要的。 </li><li>删除其后腿的螃蟹从冰桶，以防止捏它和它放置在夹层锅。 </li><li>删除先扭每一个外来的爪子。然后取出使用相同的扭转运动每条腿。 </li><li>使用rongeurs，清除覆盖口上颌骨。同时删除软maxillule。 </li><li>使用rongeurs再次，从外侧背甲后结束，并得到它的边缘上的一个良好的抓地力。打击rongeurs打破，一块背甲关闭。请一定要删除的甲壳，足以让锅铲进入开放。继续消除背甲的边缘，眼睛和上重复另一侧。 </li><li>锅铲的锥形侧面，轻轻刮去内在的软组织离背背甲的屋顶。保持接触，以避免损坏STNS甲壳锅铲。使用rongeurs割舍的甲壳已经从组织中分离出来的部分。还单独从腹侧甲壳一定要分开的任何附加的组织甲壳内收肌的一些软组织。 </li><li>打破据心，那里有两个凸出的小听骨甲壳后的背甲壳。削减前尽可能。稳定Cephalon以一个手指（“面子”），同时打破背背甲的最前部分。 </li><li>腹甲壳，一次拉出一个的下颚骨锚定到下颌骨rongeurs，扭向外逐出附加epistome的下颌骨，将它拿走。两个长的小骨与肌肉应附提取下颌骨，如果处理得当。 </li><li>离附近的眼睛，用锅铲Cephalon以双方独立的组织。随着小清扫剪刀，跨越组织尽可能层附近小骨脊层。 </li><li>靠在墙的夹层锅螃蟹和支撑保障到位（爪可用于此）。切唇（“上唇”）远离epistome确保不留下任何组织附着。使用骨切割剪刀到腹甲壳两条对角线削减删除Cephalon以。 </li><li>采取的唇齿镊和切唇（“下唇”）远离腹侧方的小骨。然后轻轻地解除嘴唇远离其余腹甲壳。用小剪刀分开组织衬背甲胃腹。当幽门壶腹是可见的，远离组织在它的两侧，直到切胃胃是从螃蟹中提取。 </li><li>仍持有的胃钳唇，其余的对手掌的胃和口盐水倒入。这扩大了胃，使得它更容易切开。从开幕式切割在上下之间通过小骨之间的壶腹的幽门权利的嘴。然后切斜通过两个贲门分支。切断三个牙齿的胃里面的提示。 </li><li>放置到一个大的，黑色的，Sylgard涂层盘内的胃降胃。牵制的编制与昆虫引脚5角和填写的菜，用冷盐水浸泡整个准备工作（见图1）。 </li></ol> 2。精细解剖<ol><li>需要优良的剥离，微清扫工具。特别是，镊子和弹簧剪刀需要以及与钨针针座。也应该有一个明确的，Sylgard涂层，培养皿和一些细铁丝引脚编制。 </li><li>最低放大倍率开始，开始牵制附近的唇，张口皮瓣。重新排列其余的引脚，使绷紧的准备和引脚任何必要的其他地区。 </li><li>小心地抬起发现皮下组织覆盖下部的准备和跨越两个白点留下一个修补皮下组织修剪。 </li><li>剥离了准备底部附近的黄，稀疏，膜状组织。 mvns是弱连接到这个组织的底面。仔细分离从黄色组织的mvns的，因为它是拉升。按照黄的组织和切旁边的大脑。 </li><li>按照现有的厚厚的脑进程，延长laterallY.这些都是commisural的神经。一旦达到连合神经年底，取出海绵，白色组织群众远离大脑的一侧已被切断。 </li><li> 离子和儿子见面的齿轮，剪去的肌肉和组织，以揭示离子和儿子的长度。 </li><li>返回发现皮下组织补丁，就把它砍下来。这应该留在周围动脉的STG开放。通过开放，切割分离的两块肌肉，侧翼的STG。提起这些肌肉的两端削权之下他们分开他们从动脉和揭露alns和活动星系核 。尽可能向上在STN躺在软骨分离的侧翼肌肉。 </li><li>切去动脉周围组织的STG mvns，DGN， DVN满足。工作DVN lvns穿过发白，细腻的组织覆盖 lvns 。 </li><li>找到超过PSN的准备底部附近的小骨。 PSN上可用于定位LVN和dlvn交界。 </li><li>下一步的dlvn找到pyn和PDN。 pyn往往包装幽门壶腹开幕。 PDN叉dlvn之间的幽门壶腹和心肺功能的幽门阀肌肉。保留一些肌肉连接到这些神经，因为他们可以很容易混淆时，准备转移到明确Sylgard菜。 </li><li>一旦所有的神经都被揭发，断绝STNS和胃组织和不必要的神经之间的任何其余的连接。小心移动STNS远离胃的其余部分，切割任何错过的连接。 </li><li>条件明确Sylgard盘剩余的胃组织在Sylgard表面擦，直到在Sylgard不再感觉太粘或干燥的质量。 Sylgard是疏水性和STNS将坚持强烈Sylgard涂层盘，如果不是以这种方式调节。 </li><li>添加一些冷盐水菜。抢钳连合神经的上端，并带来STNS明确Sylgard菜。 </li><li>引脚STNS到Sylgard。割断从连合神经脑并用的minuten引脚先关闭，以确保四个两端的齿轮。确保编制右侧检查IVN是指向远离了Sylgard。 DGN应略有退出下方STG STG是右侧。引脚其余的神经两端用细铁丝引脚下降。 </li><li>清洁干净，从STNS或任何剩余的肌肉组织。 </li><li> Desheath STG使用一个针座和一个稍微钩，罚款，钨针。小孔在STG鞘的一个角落远离胞体和使用该洞鞘层之间。小心地切割分离鞘瓣，暴露STG神经纤维和细胞组织。 </li><li>引脚鞘任何襟翼，增加无障碍的胞体和稳定的细胞内录音STG。针，确保每一根神经是绷紧和细胞外记录（见图3）间隔STNS休息。 </li></ol> 3。结果<ol><li>理想的情况下，所有的神经，应无划痕和损害，尤其是将被记录的。应的神经没有纠缠不清或扭曲。 STG应该被安排在周围的神经纤维的胡子形成细胞完好无损。完整STNS是两侧对称，看起来像一个腿 ，武器和为团长的前结束 mvns的lvns homunculus 。 </li></ol>缩写： <table border="1"><tbody><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>保留所有权利</td><td>食道神经节</td></tr><tr><td> MVN </td><td>心室神经中位数</td></tr><tr><td>离子</td><td>劣质食管神经</td></tr><tr><td>儿子</td><td>高级食管神经</td></tr><tr><td> AGN </td><td>前胃神经</td></tr><tr><td> ALN </td><td>前外侧神经</td></tr><tr><td> DGN </td><td>背胃神经</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>后路胃神经</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><tr><td> dlvn </td><td>侧脑室神经背侧支</td></tr><tr><td> IVN </td><td>劣质心室神经</td></tr></tbody></table><img src="/files/ftp_upload/1207/figure_1_90ppi.jpg" alt=“图1”/&gt; 图1：图前胃STNS近似位置剥离。 <img src="/files/ftp_upload/1207/figure_2_90ppi.jpg" alt="图2" /> 图2：在胃和浅绿色覆盖的STNS下部的各种肌肉图。 STG在每个细胞类型列出与他们来支配的肌肉。这张地图是有帮助的规划时，解剖神经，它带有一个信号从一个给定的细胞（纳迪姆实验室提供）。 <img src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" alt="图3" /> 图3：与细胞内电极和外井（黄鼠狼和布赫尔，2007年）的STNS的示意图。

<ol>
	<li>Harris-Warrick, R. M., Marder, E., Selverston, A. I., Moulins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Biological Networks: The stomatogastric nervous system. , (1992).</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>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 (892), 161-220 (1974).</li><li>Selverston, A. I., Russell, D. F., Miller, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stomatogastric+Nervous+System:+Structure+and+function+of+a+small+neural+network.">The Stomatogastric Nervous System: Structure and function of a small neural network.</a> Prog. Neurobiology. 7, 215-290 (1976).</li></ol>

STNS清扫是在执行一个电或免疫组织化学实验，或获得细胞培养细胞的第一步。不管预定的实验，总值剥离将维持不变。然而，优良的剥离可能会有所不同。重要的是要未雨绸缪的时间，你将记录从细胞和其中的神经。在STG的大多数神经元支配在肚子里至少有一个肌肉（见图2），并分类根据自己的支配目标的进程。因此，外录音需要充分验证的一个细胞内正在录制的细胞的身份。幽门节奏的频率?...

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		<div>
		<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>Black Sylgard 170</td>
<td>Other</td>
<td>Dow Corning</td>
<td>170</td>
<td>Gross Dissection</td>
</tr>
<tr>
<td>Fine Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14090-09</td>
<td>&nbsp;</td>
<tr>
<td>Insect Pins size 6</td>
<td>Surgery</td>
<td>FST</td>
<td>26000-65</td>
<td>&nbsp;</td>
<tr>
<td>Mayo Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14110-17</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Spatula</td>
<td>Surgery</td>
<td>Ward Sci.</td>
<td>15 V 4313</td>
<td>&nbsp;</td>
<tr>
<td>Rongeurs</td>
<td>Surgery</td>
<td>FST</td>
<td>16000-14</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>Surgery</td>
<td>FST</td>
<td>11021-12</td>
<td>&nbsp;</td>
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Surgery</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Fine Dissection</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Surgery</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Surgery</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Surgery</td>
<td>FST</td>
<td>15370-52</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Other</td>
<td>Fischer	</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Surgery</td>
<td>FST</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>Super fine forceps #5SF</td>
<td>Surgery</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Surgery</td>
<td>FST</td>
<td>10130-05</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Wild</td>
<td>MC3</td>
<td>Misc.</td>
</tr>
<tr>
<td>Jonah Crab (C. borealis)</td>
<td>Animal</td>
<td>Commercial Lobster</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
						
		</div>

cancer borealis stomatogastric nervous system dissection

stomatogastric节（STG）是一个很好的模型研究细胞和网络交互，因为它包含一个细胞的数量相对较少（ 约25 C。北欧化工）这是很好的特点。在STG的细胞表现出广泛的产出和胃电机行动负责。胃中含有打破了三个内部的牙齿，食物和过滤的食物，才达到的肠幽门胃磨。 STG产生两个节奏的输出来控制称为中枢模式发生器（CPGs）胃磨和幽门。 STG在每个单元格可以参加一个或这两种节奏。这些CPGs允许研究的神经调节，动态平衡，变异细胞和网络，网络的发展，和网络恢复。 解剖的stomatogastric从约拿蟹（ 癌症北欧化工 ）（STNS）神经系统两部分;总值和罚款的夹层。总值剥离整个胃解剖螃蟹。在精细剥离STNS提取使用解剖镜下微解剖工具（见图1）胃。 STNS包括STG，食道神经节（OG），并连合神经节（COG）以及腹部肌肉的神经支配。在这里，我们显示如何执行准备在STG的细胞，将细胞内和周围神经记录电生理实​​验STNS完成清扫，将用于外录音。显示我们的desheathing揭示somata和神经纤维的神经节技术，以及适当的技术，为寻找所需的的神经。

Watch this Scientific Journal Video about Cancer Borealis Stomatogastric Nervous System Dissection at JoVE.com

Cancer Borealis Stomatogastric Nervous System Dissection

stomatogastric神经系统约拿蟹（STNS）（<em&gt; C。北欧化工公司</em&gt;）可用于电生理，免疫组织化学和细胞培养的研究。 STNS提取两部分：总值和罚款的夹层。

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 ...

cancer-borealis-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Cancer Borealis Stomatogastric Nervous System Dissection

12.4K 次观看.  Brandeis. stomatogastric神经系统约拿蟹（STNS）（ C。北欧化工公司）可用于电生理，免疫组织化学和细胞培养的研究。 STNS提取两部分：总值和罚款的夹层。

视频: Cancer Borealis 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 ...

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

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 ...

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 ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

缺乏PMS2，ERCC1，Ku86，视野缺损CcOI在结肠癌进展

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

一种研究受体配体相互作用的 bw 报告系统

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

使用三维共培养系统NICO-1评估卵巢癌干细胞样细胞的血管遗传特性

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...