We would like to thank William Fletcher for animal care at the University of Ottawa. The authors would also like to thank Dr. William Milsom for teaching VT the full bilateral denervation technique at the University of British Columbia. A travel grant for this research was provided by the Faculty of Graduate and Postgraduate Studies at the University of Ottawa. This research is also supported by NSERC PGS-D scholarship to VT and Discovery Grants Program to SFP.

这两个协议符合动物护理（CCAC）加拿大议会的指引，并进行了渥太华动物护理委员会大学（协议BL-226）的批准。 1.时间差染技术:线粒体丰富的染浴<ol><li>制备1mM的MitoTracker红储液通过在94.0微升二甲基亚砜（100％DMSO）中溶解50微克。保持原液在黑暗中在-20℃下在不使用时。避免冻结/解冻循环。 </li><li>以600毫升最大音量 - （6盒3）准备深色框。用400毫升水体系（水鱼通常在举行）填充的盒子和放置一个空气石中的每个方框提供的O 2的来源。得到的金鱼（30 - 40克）和将它们放置在用400ml水和空气的石头的框。 30分钟后，添加可行线粒体富含染料，得到终浓度0.1μM和0.01％DMSO中。洗澡鱼4小时河<ol><li>如果这些控制鱼（ 即没有去神经），打开水流量在箱子上，让染料冲洗掉，恢复鱼的时间分配在协议的期限。鱼类通常回收3 - 5天。 </li></ol></li><li>恢复期后进入第3节:时间差染技术:免疫组化 。如果这些鱼是被失神经进入第2节: 全失神经支配双侧程序 。 </li></ol> 2.完全失神经支配双边程序<ol><li>获得1对学生Vannas弹簧剪刀（弯曲），1对标准模式钳直的，1对标准模式钳弯曲，2对5号镊子，一对组织拉钩，小棉球（1 - 直径2mm），并且棉签（Q提示或等同物）。 </li><li>准备麻醉水浴。首先，溶解10克苯佐卡因至100ml的最终体积99％的乙醇，以制备原料溶液。为了制备麻醉剂水浴，溶解15毫升股票苯佐卡因溶液的成30升的充气系统的水，在所要求的温度。通过将空气石连接到空气泵或中央空气线到水箱通气水中。 <ol><li>将鱼放入麻醉水浴（ 图1A）。 </li><li>一旦呼吸停止，将鱼放在一个手术表和插管它， 如图1B所示 。通过插入管在其口腔灌溉用曝气麻醉液鳃做到这一点。鳃灌溉保证了鱼与O 2和麻醉的失神经过程中足量供应。定位鱼使头部稍微向下倾斜。这使得第四鳃弓后面的区域更好的访问。 </li></ol></li><li>轻轻抬起与直标准模式钳厣并将厣和头部的内侧之间的组织牵开器。小心打开组织拉钩远离头部的鳃盖，并保持暴露鳃。 </li><li>确保麻醉的解决方案在这一过程中灌溉鳃。休息牵开手柄旁边它提供了访问所有四个鳃弓头。 </li><li>放置第四鳃弓和头部的背面之间的弯曲的标准图案镊子轻轻打开它们来创建张力附着在第四鳃弓到头部的韧带。一对5号钳创建一个小口（2 - 3毫米），通过刺穿连接鳃弓的鳃盖骨腔的背底上皮。要小心，不要去太深，因为有损害的主要血管的危险。 </li><li>与5号钳缓慢而仔细地举行了小棉球扩大切口，显露IX（舌咽）和X（迷走）神经。免费的从任何结缔组织神经通过使用5号钳，再次注意不要损伤血管。 注:鳃第九和第十金鱼的神经休息深第四鳃弓的后面，是在接近大血管送入鳃弓。 </li><li>一旦神经已经确定使用弯曲的弹簧剪刀小心切开神经按住切口弯曲形状的标准钳打开。切断神经后，轻轻收回弯钳。没有必要用缝线关闭切口因为上皮非常薄，切口通常关闭自身内24 - 48小时。删除组织牵开器。 </li><li>重复同样的步骤在头的另一侧。 </li><li>从麻醉切换鳃的灌溉新鲜充气水从麻醉中恢复的鱼。一旦鳃盖动作已恢复鱼类进入实验坦克休养至少24小时。 </li><lI&gt;在一个单独的集鱼执行"假"的过程。在"假"过程涉及刺入第四鳃弓背后的上皮不切断神经。 </li></ol> 3.时间差染技术:免疫组化<ol><li>首先，制备4％多聚甲醛（PFA）在1×磷酸盐缓冲盐水（PBS;4克PFA于96毫升的PBS）。 PFA不容易在PBS中在RT下溶解。在水浴中热溶液溶解PFA。在通风橱中执行此。一旦PFA在溶液放凉后再使用。储存在4℃下长达2周。 </li><li>前实施安乐死的鱼和提取鳃组织，放置3 - 4毫升4％PFA的成闪烁瓶中，总共8的闪烁小瓶（每鳃弓1小瓶）。此外，取一小权衡船并填写与1X PBS。这将被用于洗涤所述组织已经被切除后。置于冰上所有的解决方案。 </li><li>在可行的线粒体丰富染料曝光后实验结束后，通过与苯佐卡因过量放置在水浴安乐死的金鱼。 </li><li>使用钝钳提起厣头部的一侧，弯剪刀切断鳃鳃篮的两端。仔细拿起鳃用钝钳耙，并解除他们出鳃腔。马上洗鳃在冰冷的PBS洗涤，去除多余的苯佐卡因和血液。 </li><li>将充满用4％PFA中分开的小瓶的切除腮（小瓶每个鳃弓）和修复O / N在4℃。 </li><li>固定后，冲洗过量的PFA在1X PBS中，并放置在组织在2 ml子弹管填充用1.5ml 1％的Triton-X的上一个摇动6小时，在RT或O / N在4℃。此步骤permeabilizes组织。如果整个鳃是太大，放入2毫升管然后剪切成组织足够小，以适应管，注意不要损坏细丝部分。 <ol><li>准备一抗稀释液通过英里兴4微升每一级抗体（总共8微升）到992微升的1×PBS的原液。确保NKA（标签NKA丰富的细胞）和ZN-12（标签神经元）的主要抗体在同一主机被提出。这是很重要的，如果研究人员决定，以确定特定集成电路亚型在同一时间，他们将不得不使用在不同的宿主物种中产生初级和次级抗体。 </li><li>取出的Triton-X溶液和未经洗涤该组织中添加1:NKA单克隆抗体250稀释液（稀释于1×PBS中）（α-5），以检测NKA富含细胞和斑马鱼特定的神经元抗体（ZN-12）检测的神经纤维（第一抗体），并在室温或O / N培养在摇动器上6小时，在4℃。 </li></ol></li><li> 3分别使用的1×PBS分钟洗一次抗体的3倍。要做到这一点，通过使用移液管吸出来除去从管的第一抗体溶液。 </li><li>制备第二抗体在1:200稀释混合5微升的库存二级抗体为995微升1×PBS中。适用的二抗（的Alexa Fluor 488），孵育6小时，在RT或O / N在4℃下在振荡器上。 注:MitoTracker红色是兴奋了〜594 nm的波长，并会发出荧光红色。该被激发在〜488nm的波长和荧光绿色的二次抗体，必须使用标记的NKA和Zn-12初级抗体。 </li><li>通过洗涤组织3次，每次5分钟（如在步骤3.7中所述）除去过量的第二抗体。 </li></ol> 4.成像<ol><li>在洗涤后，装入组织上凹幻灯片整装细胞和神经纤维的共聚焦成像。要安装的组织，首先将其放置在1X PBS对一台显微镜载玻片上滴（200微升）。这确保了组织不会变干。 </li><li>分离鳃hemibranchs与弯曲微型剪刀。放置一滴的1×PBS和一滴安装介质为凹滑动。 </li><li>将分离hemibranchs到凹滑动朝上灯丝的前缘，并盖上盖玻片。轻拍盖玻片指甲油的边缘，以防止盖片从移动和位移的组织。允许将组织沉淀到凹幻灯片10的底部 - 成像前15分钟。 </li><li>对于每一个鳃弓，选择6鳃丝随机成像，每生产鳃弓六幅图像。 3微米光片 - 通过采取1使用传统的共聚焦显微镜图像的组织。 注意:任何预先存在的细胞将被贴上MitoTracker红色和阳性NKA才会出现黄色。细胞仅出现红是预先存在的IC不包含NKA。任何新增殖细胞只会是积极的NKA，只会显示为绿色。神经纤维也将出现绿色。 </li></ol>对于Ionocyte定量5.图像分析<ol><li>对于EAC已经成像ħ鳃长丝，通过滚动在Z堆叠的部分和计数的层状和丝状集成电路的数量存在，以及是否他们正在新分化，预先存在的，和/或神经支配量化集成电路和相关联的支配。 </li><li>量化每单丝或每面积长丝（毫米2）的集成电路。通过使用与用于获取图像，以勾勒出长丝包含在其中的IC进行定量的长丝的面积的薄片的软件相关联的绘图工具做到这一点。大多数共焦成像软件程序必须计算的轮廓区域的区域中的图像上的选项。使用的成像软件，它允许你这样做是为了获得该地区的选项。 </li><li>通过计算由软件，以获得每单位面积的集成电路的量度（ 例如，每平方毫米2）的面积除以计数的集成电路。 </li></ol>

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G., Zaccone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+control+of+the+gills.">Nervous control of the gills.</a> Acta Histochem. 111 (3), 207-216 (2009).</li><li>Milsom, W. K., Reid, S. G., Rantin, F. T., Sundin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrabranchial+chemoreceptors+involved+in+respiratory+reflexes+in+the+neotropical+fish,+Colossoma+macropomum+(the+tambaqui).">Extrabranchial chemoreceptors involved in respiratory reflexes in the neotropical fish, Colossoma macropomum (the tambaqui).</a> J. Exp. Biol. 205 (12), 1765-1774 (2002).</li><li>Hwang, P., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+fish+ion+regulation+and+mitochondrion-rich+cells.">New insights into fish ion regulation and mitochondrion-rich cells.</a> Comp Biochem Physiol A Mol Integr Physiol. 148 (3), 479-497 (2007).</li></ol>

The authors declare that they have no competing financial interests.

<html>时间差的染色技术可以理解离子吸收的动态调节和检查IC的时间再分配在鳃上皮的有用工具。虽然一个简单的过程，有一个数字，是至关重要的时间差染色技术的成功关键点。金鱼必须暴露于线粒体富含染料为分配在该协议的时间。短曝光将导致染料由线粒体的富细胞（ 即 ionocytes）摄取不良。期间固定，鳃组织必须迅速切下并保持在黑暗处避免光漂白。的组织，必须进行成像2周固定内处理。在双侧神经切片程序确保鱼以及麻醉;神经和血管都明确;和鱼恢复全鳃函数之前它被移动到回收罐中。 "&gt;该FW环境平衡的被动离子损失和渗透水增益14的双重挑战提出了鱼。被动离子损失的平衡，通过盐跨越这些本地化的丝状和片状上皮在那里他们可以在集成电路的主动摄取发生使与外部环境2,4,8,9,16直接接触。但是，IC的鳃位置不是一成不变的。在过去的三十年里的一些研究表明，一些FW鱼的种类，当面对与离子性和/或温度的挑战下，引入薄片向薄片1,2,4,5,17-21的更远侧区的长丝或碱鳃集成电路。这种再分配可能增加薄片的厚度可以妥协气体转移（O 2，CO 2）在整个鳃上皮22。研究人员已经用在这个手稿中描述的时差染色技术来跟踪搬迁和应急电源电子这些不同的实验条件（ 图1C）1,4,5下鳃上皮新的集成电路。 鳃，想必鳃集成电路，由IX和X颅神经支配7,23-25。这些神经同时承载传出和传入输入和从鳃上。它们分别位于第 4鳃弓背后的口腔内的背侧。神经和与该双边去神经过程可以进行的容易性的辅助功能是物种特异性的。在鳟鱼，例如，磁头的尖头和扁平解剖学允许神经在于在第 4 次鳃弓后面的单一平面下的薄层的组织。这使得神经可见的，很容易接触到的研究人员进行的失神经支配的过程。与此相反，金鱼有较短的鼻子和一个圆的头。金鱼IX和X颅神经说谎更深的背SI德第 4鳃弓占据不同的平面后腔。该取向限制了易于获得的神经，并且需要更仔细的方式来确定和切断相应的神经。去神经过程的目的是除去感觉传入和传出的输入和从鳃上。鳃弓的去神经，也可以再加上使用放射性同位素离子通量实验（ 例如，22钠）。这些技术可用于在串联研究神经输入对离子移动穿过鳃上皮的贡献。另一个限制的去神经过程是不能切断的神经从而当感觉和运动神经元之间进行区分，束可能的是两种类型的神经支配的要被删除。切断任何运动神经元可能会影响鱼的鳃鳃盖和运动。因此，在执行鳃去神经试验时，也监视后，通风是非常重要的鱼已经从过程中回收，以确保有足够的鳃运动气体和离子交换。 在这个手稿利用成年动物中所述的协议保持在12:12的光:暗周期，美联储商业食物颗粒。这些方法可以通过多种方式进行修改。首先，线粒体富含染料曝光后的恢复期可以调整，以研究人员的协议（ 例如，1，3,5，或14天）的要求。复苏的实验室线粒体丰富的染料暴露后最长期限已经14天4,5。有没有在线粒体富含染料后恢复14天的荧光的强度的显著降低。第二，利用α-5初级抗体被限制为仅识别NKA富含细胞和鳃IC的不同亚型之间没有区分。幸运的是，在金鱼已确定的大多数集成电路都是mitochondr离子富含（标签与MitoTracker）和NKA富含（标签与α-5）的细胞可能并不适用于所有的鱼类物种4,27的情况。未来的实验可以专注于通过对各种渠道，泵和换热器（ 例如，NHE，H +泵）的专门针对抗体以下具体IC亚型的时空再分配。以前的研究发现，大多数ionocytes沾上线粒体丰富染料展NKA免疫4。可以通过使用一个初级抗体对斑马鱼来源的神经元特异性抗原（ZN-12）来检测所述IC的支配。在这项研究中，α-5和Zn-12通过使用相同第二抗体（的Alexa Fluor 488），用于既检测第一抗体。这种限制是由于被提出在小鼠宿主初级抗体和由一个事实，即NKA富含细胞和神经元可以形态区分开来，即使它们发荧光相同的颜色被克服。连续染色用不同的第二抗体（ 例如，第一个α-5的Alexa Fluor 488然后ZN-12的Alexa Fluor 594），也可用于消除具有两个标记发荧光的颜色相同的问题。最后，充满双侧神经切片协议可以被修改为目标神经具体鳃弓。例如，选择性神经切片可以在第一鳃弓轻轻分开第一和第二鳃弓暴露导致第一鳃弓的鳃篮（ 图2B-E）的背底的神经进行。时间差的技术也可用于研究在幼虫斑马鱼ionocytes的分布，因为他们发展和从皮肤到鳃离子迁移过渡。

集成电路是离子型调控鱼类功能单元，并发现鳃丝上皮表面上和片晶4,6-8,10。虽然各种亚型已经描述了具有独特的功能，许多IC的特征在于一个高密度线粒体的（因此它们也被称为线粒体富细胞）和/或酶的丰度的Na + / K + ATP酶（NKA）。通常情况下，这些IC房子多种其他泵，离子通道和交换涉及离子调控（ 例如， 钠 / H +交换， 钠 / Cl -的共转运H +泵）的2,10,11。 IC的再分配和扩散的一种代偿机制是中央对维持离子稳态特别是在离子胁迫（ 如，接触离子水的差）4,8,9。 这项研究描述了一个时间差的染色TECHNI阙1，以确定鱼鳃新增殖ionocytes（集成电路）。这种技术被加上鳃弓的完整双边去神经。金鱼（ 鲫鱼 ），在这些研究中所使用的品种，非常适合学习鳃上皮细胞的增殖，因为他们有非凡的能力，以结构改造鳃2。鳃重塑是指层间细胞团时，鱼（通常保持在15 - 30℃）的生长或回缩（ILCM）被驯化冷水（&lt;15℃）或缺氧，分别为3。使用于金鱼时间差染色技术以往的研究都集中在集成电路上的鳃中鳃的上下文重塑4,5再分配，支配和增殖。加藤和金子1开发的这种新技术，研究在鳉鱼的改造和更换鳃集成电路（Fundulus heteroclitus）transferred海水（SW）淡水（FW）。在这项研究中，重点是在金鱼集成电路驯化至25℃的增殖和神经支配。 使用它表明时间差的染色技术，在鳃重塑的上下文中，金鱼保持IC的缺氧暴露和随后的含氧量正常的恢复过程中的恒定数量，但是，神经支配的细胞的百分比在整个含氧量正常恢复期5下降。有人建议超过70年前，在鱼类摄取离子机制下神经控制12。的硬骨鱼鳃由面部（Ⅶ），舌咽（Ⅸ）和迷走（X）中的神经也被称为"鳃神经"13,14支配。对斑马鱼的研究由Jonz和护士（2003）（ 斑马鱼 ）鳃神经支配表明神经支配的起源是外源性（神经纤维的细胞体是外在的鳃），以及本征（的细胞体神经纤维是内在的鳃）。同一作者还表明，鳃集成电路外在支配7。 在这项研究中，再加满双边鳃失神经时间差的染色技术被用于研究集成电路缺乏金鱼外在神经支配的增殖。满双边鳃去神经是指切断脑神经IX和X中，这两种方法都在金鱼可行的，因为它们的尺寸比较大（从30 - 200克），简化了精密的外科手术，和ionocytes使用标准免疫组织化学技术容易地鉴定。发育研究杂交瘤库，大学;在本研究中，集成电路是用一个重要线粒体特异性染料（ 例如，MitoTracker红）或针对α亚单位中的Na + / K + -ATP酶（α-5的初级抗体可视化爱荷华州，爱荷华市IA）。该协议提供了一个简单的实现方法具ð可视化和分析上的鱼鳃IC的再分配和扩散。

<table border="0" cellpadding="0" cellspacing="0" width="736">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:235px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MitoTracker Red</td>
			<td style="width:235px;">Life Technologies</td>
			<td style="width:119px;">M-7512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide</td>
			<td style="width:235px;">Sigma Alrdich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#945;-5 primary antibody</td>
			<td style="width:235px;">Develompental Hybridoma Bank</td>
			<td style="width:119px;">a5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">zn12 primary antibody</td>
			<td style="width:235px;">Develompental Hybridoma Bank</td>
			<td style="width:119px;">zn12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alexa Fluor 488 (anti mouse)</td>
			<td style="width:235px;">Life Technologies</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benzocaine</td>
			<td style="width:235px;">Sigma Alrdich</td>
			<td>E1501</td>
			<td>4-Aminobenzoic acid ethyl ester, Ethyl 4-aminobenzoate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vannas Spring Scissors</td>
			<td style="width:235px;">Fine Science Tools</td>
			<td>15000-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Pattern Forceps</td>
			<td style="width:235px;">Fine Science Tools</td>
			<td>11000-12</td>
			<td>straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Pattern Forceps</td>
			<td style="width:235px;">Fine Science Tools</td>
			<td>11001-12</td>
			<td>curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dumont No. 5 forceps</td>
			<td style="width:235px;">Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue retractor</td>
			<td style="width:235px;">Fine Science Tools</td>
			<td>17009-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:235px;">Sigma Alrdich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X</td>
			<td style="width:235px;">Sigma Alrdich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield with DAPI</td>
			<td style="width:235px;">Vector Laboratories&#160;</td>
			<td>H-1200&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

a time differential staining technique coupled with full bilateral gill denervation to study ionocytes in fish

鳃ionocytes（集成电路）的功能单元进行调控离子鱼。在成人中，它们被发现在鳃的丝状和片状上皮那里运输离子如Na +，Cl -的和Ca 2+通过多种离子通道，泵和热交换器的。的硬骨鱼鳃由面部（VI）中，舌咽（Ⅸ）和迷走（X）中的神经外在支配。在第九和第十的神经也鳃IC支配的外在来源。这里，用于研究神经支配，增殖和IC的分配两种技术进行了说明:一个时间差是染色技术和全双边鳃去神经技术。简言之，金鱼暴露于重要线粒体特异性染料（ 例如，MitoTracker红），其标签（红色荧光）预先存在的IC。鱼不是被允许恢复为3 - 5天，立即进行了全面的双边鳃去神经。后3 - 回收的5天，在克弊病收获和固定的免疫组化。然后将组织与α-5初级抗体染色（目标的Na + / K + ATP酶含有细胞）与二次抗体结合该标签的所有（新的和预先存在的）集成电路绿色。使用共焦成像，证明该预先存在的IC出现黄色（标有两个可行的线粒体特异性染料和α-5）和新的IC出现绿（标有α-5只）。串联使用两种技术可用于研究在鳃丝IC的神经支配，增殖和分发时鱼暴露于环境挑战。

图1示出了外科手术表成立（ 图1A），鱼的手术过程中的放置（ 图1B）和时间差染色技术（ 图1C）的三个最重要的步骤。在步骤1，将鱼保持30分钟以充分充气水浴在25℃下在黑暗中。在30分钟期间，研究者可以准备在DMSO线粒体富含染料等份该步骤2（ 图1C）中加入到水中。步骤2中的潜伏期允许线粒体富含染料从水进入线粒体富细胞（ 即集成电路）的摄取。这种鱼可以那么无论进行全面的双边失神经过程或其中鱼是麻醉是假的程序和操作的鳃盖，但神经保持不变。对设立在第3步代表鱼有荣提供流水恢复室经过疗法双侧全失神经支配或"假"的过程。 恢复期后，将鱼被安乐死并鳃切除并固定用于免疫组织化学。集成电路上经历了一个"假"过程的鱼的鳃丝的总体分布和神经支配描绘在图2的IC是存在于丝状上皮以及在层间的区域。 图2A示出的基标记线粒体富含染料预先存在的IC（ 即，这些集成电路存在的去神经/假程序进行之前）。 图2B示出的神经纤维支配预先存在的和新形成的集成电路的丝状的（确定的免疫反应性NKA）和板层上皮细胞。最后，将两个图像的合并（ 图2C）清楚地表明预先存在的集成电路（出现黄色）和新的IC（显示为绿色）。图2D是集成电路量化的代表性曲线图在图2A-C中所描绘的长丝。在这个特定的长丝，似乎有每平方毫米新近增殖IC的更大数目比预先存在的IC（N = 1）。以除去外在鳃神经支配的来源，所述第九和第十颅神经（外在神经支配）被切断。 图3A示出了鳃丝后鳃腔的背侧区域和覆盖所述神经上皮被除去，以暴露九和十颅神经（以罗马数字表示）从两个主要神经树干跨越到支配所有四个拱（ 图3A）;鳃弓的编号为1到4 图3B-E说明第一鳃弓的选择性失神经支配。第一鳃弓由两个IX和X颅神经（ 图3B），它可以在不影响支配到静止ø去除神经支配f显示鳃弓（ 图3C-E）。全失神经双边导致外源性神经支配逐渐丧失的鳃丝（ 图4A-C）。控制鱼表现出明显的神经束横跨长丝（ 图4A）的长度。满双边去神经导致外在神经支配恢复后的2天（ 图4B）的一些损失。外在神经支配的进一步消失注意到后从去神经（ 图4C）5天恢复。任何剩余的神经支配到集成电路5天后回收的推测来源于神经与细胞体的鳃丝内（特性支配; 图4C）。 <img alt="图1" src="/files/ftp_upload/52548/52548fig1highres.jpg" width="600" /> 图1.实验成立的步骤中所用的去神经过程和顺序时差染色技术。 （A）手术台设立失神经支配的过程显示麻醉和恢复坦克。放置的麻醉，气管插管的鱼（B）为例，在该时间差染色技术使用的（C）的关键步骤。在步骤1，将鱼放置在一个温度控制的，静态的充气水浴中30分钟。线粒体富含染料在步骤2加入到0.1μM的终浓度和鱼是允许在最少4小时的溶液洗澡。水流重新启动在步骤3在这一点鱼经过鳃的一个全双边去神经或假手术。 <a href="https://www.jove.com/files/ftp_upload/52548/52548fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52548/52548fig2highres.jpg" width="600"<html> /&gt; 图2.光显微照片表示在金鱼全双边去神经后第2天的时间微分染色技术（驯化至25℃）。 （A）中预先存在的ionocytes（集成电路;箭头）的分布。在单个鳃丝是由线粒体富含染料染色显示（B）的集成电路的分布（新的和预先存在的）和鳃神经（虚线箭头）通过与α-5和Zn-12抗体，染色分别显露。箭头指示的IC（C）的 （A）和（B）中的重叠区分预先存在的IC。;从新近增殖的IC（出现黄由箭头指示）（出现绿色;由箭头指示）。插入（C）是一种神经支配预先存在ionocyte的倍率。（D）中的IC量化为在面板（AC）中所示的长丝的代表性曲线图。面板N = 1比例尺（C）为50微米，并适用于所有的面板。/ftp_upload/52548/52548fig2highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52548/52548fig3highres.jpg" width="700" /> 图3代表性图像描绘鳃神经支配的各个阶段。 （A）背口腔显示IX和X颅神经支配的所有4鳃弓的看法。鳃弓的编号1-4。鳃丝和组织覆盖被拆除的神经，以更好地可视化的支配。（B）第一届和第二鳃弓分离揭示感觉器官和IX和X颅神经的分支支配1日鳃牌坊。图像显示用切断IX和X颅神经的分支1日鳃弓的选择性失神经支配（CE）序列。对于示意图重演示硬骨鱼鳃支配，请参阅图1米尔森等[26]在图像中的白线是从显微镜灯水中的倒影。它们不限定任何形态结构。面板比例尺（E）为4毫米，适用于所有的面板。 <a href="https://www.jove.com/files/ftp_upload/52548/52548fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52548/52548fig4highres.jpg" width="700" /> 图4.光显微照片描绘ionocytes的分布和神经支配的驯化至25℃。Ionocytes（由箭头头部指出） 一个金鱼的1次鳃弓的单个长丝进行染色与α-5抗体和神经（指示BŸ箭头）沾满锌-12抗体。控制鱼呈现出中央神经束从第九和第十神经（外源性神经支配）提供广泛的层状分支想必发起的（A）鳃丝。一些国家表示了ionocytes也都支配（插入）。（B）在鳃丝全失神经双边术后第2天。有减少中央神经束，而层状支配出现基本完好。（C）鳃丝全失神经支配的双边表明外在神经支配是基本上不存在后5天。定性的分析表明，完整的双边去神经引起的外在鳃神经支配的劣化，同时维持神经与鳃丝内的细胞体（固有神经支配）通过灯丝和成薄片产生的神经网络。 <a href="https://www.jove.com/files/ftp_upload/52548/52548fig4highres.jpg" target="_blank">请</a>点击此处查看该图的放大版本。

Watch this Scientific Journal Video about A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish at JoVE.com

A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish

This manuscript describes a protocol to track the re-distribution of branchial ionocytes and their innervation using a time differential staining technique coupled with full bilateral gill denervation.

一时间差别染色技术再加上双边完全失神经支配吉尔在鱼类研究Ionocytes

Branchial ionocytes (ICs) are the functional units for ionic regulation in fish. In adults, they are found on the filamental and lamellar epithelia of the ...

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Research

JoVE Journal

Developmental Biology

8.3K 次观看.  University of Ottawa. This manuscript describes a protocol to track the re-distribution of branchial ionocytes and their innervation using a time differential staining technique coupled with full bilateral gill denervation. 

视频: A Time Differential Staining Technique Coupled with Full Bilateral Gill Denervation to Study Ionocytes in Fish

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

在小鸡学习感应信号与候选因子浸泡珠结合防渗屏障中的应用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

一时间差别染色技术再加上双边完全失神经支配吉尔在鱼类研究Ionocytes

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