我感谢Alex L. Kolodkin，因为我在他的实验室里学到了这个填写的准备方案。我也非常感谢杨宏宏的技术援助。本研究得到NRF-2013R1A1A4A01011329（SJ）的支持。

准备<ol><li>通过加入0.5g牛血清白蛋白（BSA）和0.5mL叔辛基苯氧基聚乙氧基乙醇（参见材料表），向具有叔辛基苯氧基聚乙氧基乙醇（PBT）溶液的500ml磷酸盐缓冲盐水（PBS）中制备500mL的1X PBS并搅拌至少30分钟。储存于4°C。使用时比较新鲜，并将溶液储存在干净的瓶子里。 </li><li>通过向6.5mL去离子水中加入2.5mL 16％的多聚甲醛溶液和1mL的10x PBS，制成10mL的4％多聚甲醛。储存于4°C并在一周内使用。 小心:避免皮肤接触多聚甲醛溶液，因为它具有高毒性。 </li><li>通过每1mL DMSO溶解0.1g X-Gal底物制备X-Gal底物（10％w / v X-Gal在二甲基亚砜（DMSO）中）。在-20°C储存在100μL等分试样中。 </li><li>使用10mM磷酸盐缓冲液（pH7.2），150mM NaCl，1mM MgCl 2，3μm制备X-Gal染色溶液MK 4 [FeII（CN） 6 ]，3mM K 3 [FeIII（CN） 6 ]和0.3％叔辛基苯氧基聚乙氧基乙醇。在黑暗中储存于4°C。 </li><li>用去离子水稀释30％的过氧化氢1:10制成3％过氧化氢溶液。 </li><li>通过将1片（10mg）DAB完全溶解在35mL新鲜PBT溶液中来制备3,3&#39;-二氨基联苯胺（DAB）溶液。通过0.2μm注射过滤器过滤，并在-20°C下保存在910μL等分试样中。 小心:将所有DAB废物处理在漂白剂中以摧毁民建联。 </li><li>用苹果汁制作蛋盘。向2L烧瓶中加入35g琼脂和1L去离子水。向1升烧瓶中加入33g蔗糖，2g tegosept和375mL苹果汁。通过高压灭菌30分钟使其熔化。允许两种溶液冷却至约60℃。通过搅拌结合并混合这些溶液。每60毫升培养皿倒入约11毫升的组合溶液。 </li><li>通过混合面包酵母制作酵母糊st进入去离子水（1:0.8比例）并储存在4℃。 </li></ol>胚胎收集（第1天） <ol><li>收集年轻的雌雄和雄蝇（5日以下），并保持1-2天，将它们保持在一个塑料烧杯笼中，里面装有少量的酵母糊（60毫米x 15毫米）。 </li><li>为了最佳收集晚期16期胚胎，在5点左右的时间内建立一个带有苍蝇（&gt; 50）的瓶笼，让它们在25℃下孵育3小时。 </li><li>将苍蝇转移到新鲜的食物瓶中。 </li><li>用盖子关闭蛋盘，并在25℃下在潮湿的培养箱（约70％湿度）中将其倒置过夜。 </li></ol> 3.免疫染色胚胎的制备（第2天） <ol><li>在上午10点20分左右将蛋白质加入1.8毫升PBT溶液中，并用棉签将其从板上松开，同时倾斜。 注意:用cott轻轻地捣碎酵母糊在拭子上，溶解它，以防发现大量的胚胎。开始收集胚胎〜固定前约40分钟（约11:00 AM）。 </li><li>将胚胎从蛋板转移到1.5 mL微量管中，使用1 mL移液管吸头。 </li><li>允许胚胎沉淀并尽可能吸出PBT溶液。用1 mL PBT溶液冲洗胚胎两次。尽可能吸出PBT溶液。 </li><li>向管中注入1 mL 50％的漂白剂（ 即稀释在去离子水中），并通过在室温下（RT）将它们培养在营养器上3分钟来使胚胎脱钙。 注意:这一步不会杀死被破坏的胚胎。 </li><li>让脱钙的胚胎沉淀，尽可能吸出50％的漂白剂，并用1 mL的PBT溶液冲洗胚胎3次。 </li><li>加入0.5mL庚烷，随后在约11:00 AM的室温下向管中加入0.5mL的4％多聚甲醛溶液。 </li><li>孵化在一个养分器上的胚胎在室温下15分钟。 </li><li>取出4％多聚甲醛溶液（底层）。 </li><li>加入0.5 mL 100％甲醇，通过剧烈摇动管30秒，使胚胎变细。 </li><li>除去庚烷（ 即顶层）。 </li><li>加入0.5 mL 100％甲醇，剧烈摇动管10秒。 </li><li>通过点击管子尽可能地吸出甲醇使胚胎沉降。用1 mL 100％甲醇冲洗胚胎，摇动不到10秒。 注意:长期暴露于甲醇会破坏β-Gal活性。 </li><li>取出甲醇并用1 mL PBT溶液冲洗脱皮胚胎3次。 </li></ol> 4.使用LacZ染色对胚胎进行基因分型（第2天） <ol><li>将1mL PBT溶液加入到管中，并将管在37℃的热块或水浴中孵育15分钟。 </li><li>孵育期间，放置X-Gal染色溶液（1 mL / tu在65℃在水浴中，直至变得多云。在37°C水浴中孵育。 </li><li>吸出PBT溶液，并向管中加入1 mL X-Gal染色溶液和20μLX-Gal底物。 </li><li>在37℃下孵育管，使其具有营养作用，直到蓝色沉淀物明显。 注意:第2和第3个蓝色平衡器的孵育时间一般在2-4小时之内。 </li><li>取出X-Gal染色溶液，用1 mL RT PBT溶液冲洗胚胎3次。 </li><li>使用针头或镊子，用光解剖显微镜（1.6X-2.5X目标）手工分选所需基因型的胚胎（ 即未染色的白色胚胎）。 注意:由于一些蓝色平衡剂，如CyO，肌动蛋白-LacZ和TM3，肌动蛋白 -LacZ，携带在肌动蛋白启动子控制下表达细菌β-Gal（LacZ）的转基因构建体，以无处不在的方式染成蓝色的胚胎含有蓝色平衡染色体的一或两个拷贝。因此，未染色的白色胚胎对于所需的致死等位基因是纯合的。 </li></ol> 5.用抗Fasciclin II抗体对胚胎进行免疫染色（第2-3天） <ol><li>使用1 mL移液管吸头收集并转移所需基因型胚胎（ 即未染色的白色胚胎）至0.5 mL微量管。 注意:在这个步骤中，胚胎可以根据形态学标准大致分期，包括角质层的分割模式和头尾的结构9 。在头部退化期间，蛋放置后10至16小时，胚胎经历了最前段9的显着减少。 </li><li>用0.4mL PBT溶液洗涤胚胎一次，并加入0.3mL封闭溶液（5％正常山羊血清和5％DMSO在PBT溶液中）。 </li><li>在室温下将营养物阻断15分钟。 </li><li&gt;加入75μL抗Fasciclin II抗体，并在室温下孵育具有章动作用的管。 </li><li>用0.4mL PBT溶液洗涤胚胎4次，每次洗涤至少20分钟。 </li><li>加入含有山羊抗小鼠HRP抗体（2μg/ mL）的0.3mL封闭溶液（PBT溶液中的5％正常山羊血清），并将管子在室温下孵育过夜。 </li><li>用0.4mL PBT溶液洗涤胚胎6次，每次洗涤至少20分钟。 </li><li>向管中加入0.3mL DAB溶液。 小心:戴上手套，将所有DAB废物/管/尖端放入漂白剂中。 </li><li>加入2μL3％过氧化氢，并在RT中将管子在黑暗中孵化，直到产生所需量的沉淀物。 注意:1D4免疫组化的培养时间一般在0.5-1小时。 </li><li>用0.4mL PBT溶液洗涤胚胎4次。 小心:取出DAB溶液，然后用移液管和二极管清洗前两次洗涤液把它们漂白掉。 </li><li>向管中加入0.2 mL安装的70％甘油/ PBS溶液，并在室温或4°C储存。 注意:保持管远离光线。通过将胚胎置于90％甘油/ PBS溶液10中可获得更清楚的制剂。然而，比在70％甘油/ PBS溶液10中清除的那些更容易解剖在90％甘油/ PBS溶液中清除的胚胎。 </li></ol> 6.分期，分解，安装和成像胚胎（第4天） <ol><li>为了分期，将用70％甘油/ PBS平衡的胚胎转移到载玻片上。 </li><li>收集具有收敛于彼此接触或/并且具有细分为三个带的中肠的腹部节段7（A7），A8和A9 ISN神经的晚期16期胚胎。 注意:1D4抗体染色的胚胎可以基于ISN和ISNb运动轴突的投影图案进行分期。退出CNS，A7，A8和A9 ISN之后晚期-16胚胎的rves会聚到彼此接触，随后发散到远离背部肌肉（ 图1A中的箭头）。然而，在中期16期胚胎中，A7 ISN神经与A8 / A9神经（ 图1B中的方括号）平行延伸。此外，两个半身中的A6 ISNb神经（垂直于腹侧肌）的投影轴与CNS纵向轴突束的后端（箭头和图1C中的一条线）大致对齐。这些形态学标准在不同的基因型之间有所不同。或者，胚胎可以基于肠道形态9,11进行分期。在阶段17之前，中肠具有心形形状，而中肠在阶段17 12细分为三个带。 </li><li> 解剖胚胎。 <ol><li>使用1米L注射器，将每个胚胎移出甘油滴，并将胚胎腹面朝上。在身体长度的1/4处切除前部，并且没有运动轴突的最后部区域。 </li><li>使用针头探针，将终端胚胎移出甘油滴，滚动以将其背面朝上定位，并将其水平放置在野外。 注意:当胚胎移出甘油滴时，与胚胎相关的一点甘油使其粘稠。 </li><li>使用单个非常精细的钨针13沿背侧中线切割胚胎。在胚胎后端做一个小切口，然后继续将背部中线向前切割;相反方向的切割也很好。 </li><li>使用针头探针，将中线切割的胚胎移入甘油滴，将其背面放置，并通过在腹侧（对角线）方向上展开每个体壁（2），将肠道从体壁分离.5X目标）。 注意:确保背部中线完全切割，导致左右两侧体壁之间完全分开。 </li><li>仔细将中线切割的胚胎从甘油滴中移出，然后使用细针探针（2.5X物镜）将身体壁上的两个皮瓣放在玻璃片上。 </li><li>使用非常精细的钨针，通过将其推向侧面（5X物镜）从分离的胚胎中去除内部器官。 注意:另一个类似的解剖程序的更详细的描述可以在网站上找到。 </li></ol></li><li>为了安装，加入2-3μL70％甘油/ PBS溶液清洁，解剖的胚胎，并使用细针探针将其转移到新的载玻片上，将8μL70％甘油/ PBS溶液涂抹在其上中心（2.5X目标）。 </li><li>穿上盖玻片（18毫米x 18毫米）。用常规指甲油（也可以）密封盖玻片的边缘清晰或有色是好的）。 </li><li>根据制造商的说明，在差分干涉对比（DIC）光学显微镜下以高分辨率（20X，40X和63X浸油物镜）拍摄图像。 注意:更好的可视化和表型特征，特别是对于ISNb运动轴突，可以通过使用63X油浸物镜来生产。 </li></ol>

<ol>
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Rev. Neurosci. 13 (9), 605-618 (2012).</li></ol>

笔者宣称他没有竞争的经济利益。

运动轴突指导缺陷的细节通过DAB染色的胚胎的切片制备比通过荧光标记的激光扫描共焦显微镜更快，更准确。因此，固定和1D4染色的胚胎的切片制备最适合于指导提示分子的功能表征。指导线索包括网络，狭缝，信号素（Semas）和水母以及他们的同源受体在蠕虫，苍蝇和脊椎动物中具有进化上保守的作用。在作为狭缝受体的迂回（Robo）家族分子中， 果蝇 Robo在CNS 6,21中首先被确定为调解排斥引导功能。 Semas是一系列与plexin相关的受体复合物结合的指导分子。 Semas的轴突引导功能在蝗虫CNS 14中首次发现。随后，1号跨膜信号素Sema-1a在果蝇中被广泛表征 。 Sema-1a不仅作为plexin A的配体，而且作为未知配体的受体起着重要的作用，在胚胎神经发育期间的特定选择点，在轴突神经轴突排斥15,16,17,18（ 图2C ）。 当前协议可能会出现一些常见的故障排除步骤。首先，在步骤3.9-3.13中，如果在甲醇固定化过程中使用太多或太小的胚胎，则胚胎可以以低效率进行脱皮化。在这种情况下，建议在步骤3.5中使用40-80微升固定胚胎进行脱石化。第二，在ps 3.9-3.13，由于用作固定剂的甲醇会破坏β-Gal活性，所以在长时间暴露于甲醇或不完全庚烷除去的情况下，可能会发生弱X-Gal染色。在这种情况下，最好尽可能快地执行步骤3.9-3.13中使用甲醇的脱铁程序。此外，溶解在甲醇中的残余庚烷可能会干扰染色;因此，最好在步骤3.11-3.13中除去尽可能多的甲醇。 目前的切片制备方案可以应用于功能丧失和功能增强遗传筛选，以鉴定涉及运动轴索寻路和靶标识别的新基因。然而，该方案的缺点在于解剖固定的胚胎以产生切片制剂是非常耗时的。为了克服这个问题，可以在1D4染色的整体制备中监测轴突突变的表型表征ryos，尽管这种方法提供了周边轴突投影的低分辨率可视化。然而，全基准备方案已被证明是筛选轴突指导基因的有效方法4 。最近开发了一种使用荧光标记胚胎活体解剖的替代方案进行系统筛选。活体解剖方案允许相对快速的表型分析准备11 。然而，这个方案对于早于17期初期的胚胎来说是不够的。 本文所述的切片制备方案可用于检测不同抗体的蛋白质表达模式。此外，该方案还可以应用于各种发育阶段的果蝇胚胎。

胚胎发育期间运动轴突和目标肌肉之间的精确和选择性联系对于果蝇幼虫的正常运动是必不可少的 。每个腹部疾病A2-A7中的30个肌纤维的胚胎图案是由阶段16 1建立的 。在腹侧神经线中产生的36只运动神经元将其轴突延伸到外周以支配特定的目标肌肉2 。通过抗体（小鼠单克隆抗体1D4）3,4免疫组织化学可以观察到运动轴索寻路和靶标识别。在野生型胚胎中的运动轴突投影图案的多个图像可在网络5上获得 。 1D4抗体标记胚胎中枢神经系统（CNS）中线每侧的所有运动轴突和三个纵向轴突束</s， 6 （ 图1C和图2A ）。因此，FasII抗体的免疫组化提供了一个强大的工具，用于识别神经肌肉连接所需的基因，以证明运动轴突指导和靶标识别的分子机制。 在每个腹部A2-A7中，运动轴突突出并选择性地成形为两个主要神经分支，节段神经（SN）和节段间神经（ISN） 2,4和一个小神经分支，横向神经（TN ） 7 。 SN选择性地消弧以产生称为SNa和SNc的两个神经分支，而ISN分裂成称为ISN，ISNb和ISNd 2,4的三个神经分支。其中，ISN，ISNb和SNa运动轴突当晚期16期胚胎用FasII抗体染色时，投影图案最精确地可视化，并被切片（ 图1C和图2A ）。 ISN运动神经元延伸其轴突以支配1,2,3,4,9,10,11,18,19和20,2,4（ 图2A ）的背部肌肉。 ISNb运动神经元支配腹侧肌6,7,12,13,14,28和30,24（ 图2A和2B）。 SNa神经分支突出支配5,8,21,22,23和24,2,4（ 图2A ）的侧肌。由两个运动轴突组成的TN沿着节段边界向内侧投射以支配肌肉25，并且在侧向双极树突状神经元（LBD）中突触周边7 （ 图2A ）。这些目标肌肉神经支配不仅需要在特定选择点的运动轴突的选择性阻塞，而且还需要靶向肌肉识别。此外，在ISN和SNa途径中都发现了一些作为中间靶点的推定的中胚层路标细胞，但不是沿着ISNb途径4 。这可能表明与ISN和SNa运动轴突指导相比，ISNb运动轴索寻路可以以明显的方式进行调节，并且还表明外周运动轴突指导提供了一个有吸引力的实验模型来研究单个指导提示的差异或保守作用分子8 。 这项工作提出了一种标准方法，可视化果蝇中胚胎运动神经元的轴突投影模式。所描述的方案包括如何解剖用1D4a染色的固定胚胎在3,3&#39;-二氨基联苯胺（DAB）中加工用于切片制剂。固定胚胎的扁平制剂的一个关键优点是周边轴突突起和肌肉模式的更好的可视化。此外，这项工作还显示了如何使用LacZ染色法将固定的胚胎基因型分类为所需的突变体胚胎。

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Bovine Serum Albumin</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">A7906</td>
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			<td height="40" style="height:40px;width:284px;">Triton X-100</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">X100</td>
			<td style="width:228px;">t-Octylphenoxypolyethoxyethanol</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:284px;">16% Paraformaldehyde Solution</td>
			<td style="width:144px;">Ted Pella</td>
			<td style="width:131px;">18505</td>
			<td style="width:228px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Sodium Chloride</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">S5886</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Potassium Chloride</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">P5405</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Sodium Phosphate Dibasic</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">30435</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Sodium Phosphate Monobasic</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">71500</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:284px;">X-Gal Substrate</td>
			<td style="width:144px;">US Biological</td>
			<td style="width:131px;">X1000</td>
			<td style="width:228px;">X-Gal (5-Bromo-4-chloro-3-indolyl-b-D-galactoside galactopyranoside)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Dimethyl Sulfxide</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">D4540</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Magnesium Chloride</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">M8266</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:284px;">Potassium hexacyanoferrate(II) trihydrate</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">P9387</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Potassium hexacyanoferrate(III)</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">244023</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Hydrogen Peroxide</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">216763</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">3,3&#39;-diaminobenzidine Tetrahydrochloride</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">D5905</td>
			<td style="width:228px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Agar</td>
			<td style="width:144px;">US Biological</td>
			<td style="width:131px;">A0930</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Sucrose</td>
			<td style="width:144px;">Fisher Scientific</td>
			<td style="width:131px;">S5-3</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Tegosept (Methy 4-Hydroxybenzoate)</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">H5501</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Culture Dish (60 mm)</td>
			<td style="width:144px;">Corning</td>
			<td style="width:131px;">430166</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:284px;">Tricon Beaker</td>
			<td style="width:144px;">Simport</td>
			<td style="width:131px;">B700-100</td>
			<td style="width:228px;">This is used to make a plastic beaker cage for embryo collection.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:284px;">Yeast</td>
			<td style="width:144px;">Societe Industrielle Lesaffre</td>
			<td style="width:131px;">Saf Instant Yeast Red</td>
			<td style="width:228px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:284px;">Cotton Swab (Wooden Single Tip Cotton PK100)</td>
			<td style="width:144px;">VWR</td>
			<td style="width:131px;">14220-263</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Eppendorf Tube (1.5 ml)</td>
			<td style="width:144px;">Sarstedt</td>
			<td style="width:131px;">#72.690</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Bleach</td>
			<td style="width:144px;">The Clorox Company</td>
			<td style="width:131px;">Clorox</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Heptane</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">246654</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Methanol</td>
			<td style="width:144px;">J.T. Baker</td>
			<td style="width:131px;">UN1230</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Normal Goat Serum</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:131px;">16210-064</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:284px;">Anti-FasciculinII Antibody</td>
			<td style="width:144px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:131px;">1D4 anti-Fasciclin II</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:284px;">Goat Anti-mouse-HRP Antibody</td>
			<td style="width:144px;">Jackson Immunoresearch</td>
			<td style="width:131px;">115-006-068</td>
			<td style="width:228px;">AffiniPure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG+IgM (H+L) 
			(min X Hu, Bov, Hrs Sr Prot</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Glycerol</td>
			<td style="width:144px;">Sigma-Aldrich</td>
			<td style="width:131px;">G9012</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Slide Glass</td>
			<td style="width:144px;">Duran Group</td>
			<td style="width:131px;">235501403</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Coverslip</td>
			<td style="width:144px;">Duran Group</td>
			<td style="width:131px;">235503104</td>
			<td style="width:228px;">18 x 18 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:284px;">1 ml Syringe</td>
			<td style="width:144px;">Becton Dickinson Medical(s)</td>
			<td style="width:131px;">301321</td>
			<td style="width:228px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:284px;">Tungsten Needle</td>
			<td style="width:144px;">Ted Pella</td>
			<td style="width:131px;">#27-11</td>
			<td style="width:228px;">Tungsten Wire, &#248;0.13mm/6.1m (&#248;.005&#34;/20 ft.)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:284px;">Nutator (Mini twister)</td>
			<td style="width:144px;">Korean Science</td>
			<td style="width:131px;">KO.VS-96TWS</td>
			<td style="width:228px;">Alternatively, BD Clay Adams Brand Nutator (BD 421125)</td>
		</tr>
	</tbody>
</table>

visualization of the axonal projection pattern of embryonic motor neurons in drosophila

功能性神经肌肉回路的建立依赖于发育中的运动轴突和目标肌肉之间的精确连接。运动神经元通过响应大量来自周围细胞外环境的轴突指导线索，延长生长锥体以沿着特定途径导航。生长锥目标识别也在神经肌肉特异性中起关键作用。这项工作提出了一个标准的免疫组织化学方案，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。该方案包括几个关键步骤，包括基因分型程序，以排序所需的突变体胚胎;免疫染色程序，用fasciclin II（FasII）抗体标记胚胎;和解剖程序，从固定胚胎产生鱼片制剂。周边的运动轴突突出物和肌肉图案在圆形胚胎的扁平制剂中比在wh中更好地可视化油籽胚胎。因此，用FasII抗体染色的固定胚胎的切片制备为表征运动轴突寻路和靶标识别所需的基因提供了有力的工具，也可以应用于功能丧失功能遗传屏障。

神经发育期间运动轴突和目标肌肉之间的精确连接取决于在特定选择点的选择性轴突 - 轴突排斥和目标识别。在果蝇中 ，运动轴突之间的选择性排斥部分由第1类和第2类信号素（Semas）（包括Sema-1a，Sema-2a和Sema-2b）8,14,15,16,17,18的组合作用。因此， Sema-1a功能的丧失常常导致ISNb轴突在特定选择点处失效，导致其表现出异常浓厚的形态（ 图2C中的箭头）。在野生型胚胎中，至少有7只运动神经元延伸并使其轴突形成一个I形成的轴突SNb神经分支（ 图2A ）。当ISNb神经分支到达位于肌肉6和7之间的选择点时，两个轴突选择性地从主ISNb束起始并随后使肌肉6和7支配（ 图2B ）。在位于肌肉6,13和30之间的下一个选择点，另外三个运动轴突选择性地缩小以支配肌肉13,14和30（ 图2B ）。剩余的两个轴突向背侧延伸以到达肌肉12边缘附近的最后一个选择点，从而在相反方向上支配肌肉12（ 图2B ）。这些结果清楚地表明，切片制备方案提供了一个有用的工具来研究运动轴突指导所需的基因。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55830/55830fig1.jpg" /> <str图1:基于ISN和ISNb运动轴突的突出图案的胚胎分期。 （ A ）在晚期-16野生型胚胎中，A7，A8和A9 ISN神经（箭头）收敛于彼此接触（箭头）。前路留下，腹侧下垂。比例尺= 15μm。 （ B ）在中期16型野生型胚胎中，A7 ISN神经与A8 / A9神经（方括号）平行延伸。箭头表示A7，A8和A9 ISN神经。前路留下，腹侧下垂。比例尺=15μm。 （ C ）用FasII抗体染色的晚期16型野生型胚胎的片状制剂。左右A6 ISNb神经的突出轴（箭头）大致与CNS纵轴索束（黑线）的后端对齐。腹部A1-A7标记在顶部。前方留下比例尺= 15μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55830/55830fig1large.jpg" target="_blank">恳求</a>请点击这里查看此图的较大版本。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55830/55830fig2.jpg" /> 图2:野生型和Sema-1a突变体胚胎中的运动轴突投影模式。 （ A - C ）用FasII抗体染色的晚期-16胚胎的切片制剂。前路留下，腹侧下垂。示意图显示每个面板中表示的轴突投影图案。刻度棒=15μm。 （ A ）野生型胚胎中两个连续腹部血液透析比A3，A4的明场照片。 CNS中存在三个双侧对称的纵向轴突束。 ISNb，SNa，ISN和TN运动轴突的正常神经支配模式由周围的框和箭头指示。支配阿拉伯肌肉的侧向双极枝晶神经元（LBD）是指示物由箭头编辑。在原理图中，提出了ISNb（棕色），SNa（蓝色），ISN（绿色）和TN（黄色）运动神经元及其目标肌肉的神经分支和神经支配模式。 CNS中这些运动神经元的细胞体位置也以自己的颜色显示。 （ B ）在野生型胚胎中，ISNb轴突投射到腹外侧肌肉，其编号，并在特定选择点（DIC图像中的箭头）支配它们。 （ C ）在Sema-1a功能缺失突变体中，ISNb轴突在肌肉6和13（DIC图像中的箭头）之间经常不能彼此消失。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55830/55830fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila at JoVE.com

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

这项工作详细描述了一个标准的免疫组织化学方法，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。用FasII抗体染色的固定胚胎的切片制备提供了一个强大的工具，用于表征神经发育期间运动轴索寻路和靶标识别所需的基因。

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

visualization-axonal-projection-pattern-embryonic-motor-neurons

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

7.7K 次观看.  Chonbuk National University. 这项工作详细描述了一个标准的免疫组织化学方法，可视化晚期16 号果蝇黑色素瘤胚胎的运动神经元投影。用FasII抗体染色的固定胚胎的切片制备提供了一个强大的工具，用于表征神经发育期间运动轴索寻路和靶标识别所需的基因。 

视频: Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

运动神经元细胞外识别的肌肉运动游泳池<em&gt;海兔夜蛾</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1.
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students&#8217; practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish&#8217;s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

小龙虾的游泳足系统:实用指南为神经线及电机模式外记录的解剖

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

小鼠膈运动神经元逆行神经解剖学追踪的研究

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...