笔者想感谢路易斯·雷查德博士为TrkA的taulacZ小鼠和安妮特·马库斯博士有见地的讨论和建议。这项工作是从伯克基金会以及白厅基金研究资助2010-08-61，从翅膀生命基金会（WFL-US-028/14）研究经费支持的启动资金，从授予ZB1-1102-1克里斯托弗＆达娜里夫基金会和赠款1R01EY022409和3R01EY022409-01S1从国家眼科研究所，为JZ。 KJO是史密斯的家伙。

注:所有程序符合美国国立卫生研究院指南实验动物的使用和护理。动物方案得到批准IACUC威尔康乃尔医学院。 1.组织准备<ol><li>颈椎脱位安乐死15定时女性怀孕。解剖胚胎E16 - 从定时怀孕的女性和地点胚胎E18胚胎逐个在6孔培养皿的孔中，填充有冷的磷酸盐缓冲盐水（PBS）。 </li><li>在冷PBS冲洗胚胎。 </li><li>在随后的基因分型的预制备的蛋白酶K溶液中取出胚胎和地点的羊膜的一小部分。或者，如果羊膜丢失，除去的胚胎尾巴尖的一小部分，并将其放置在蛋白酶K溶液</li><li>戳孔进入皮肤四周用昆虫针（至少100每边）的胚胎。 </li><li>删除PBS慢慢补充新鲜2％多聚甲醛（PFA），pH为7.4，向井。 </li><li>轻轻摇动2小时，4℃。 </li><li>在PBS中漂洗两次胚胎并在PBS中储存于4℃直至染色。 </li></ol> 2.基因分型<ol><li>根据制造商的说明浸入羊膜或胚胎尾巴在蛋白酶K混合物。 </li><li>孵育在56℃下进行10分钟。 </li><li>使用市售的DNA纯化试剂盒提取DNA。 </li><li>取0.5微升的总200微升纯化的基因组DNA溶液，用0.5微升每个基因特异性义和反义引物（10μM）的，2微升dNTP混合物结合它（2.5毫的dATP，dCTP，dGTP和dTTP的） ，0.2微升Taq聚合酶（5U /微升）和2微升的PCR缓冲液（10倍），加入的H 2 O来的20微升的总体积。 注:引物序列如下（ 见表1）: TrkA的:TrkA_F:GCG GGC GCC GCC GCG ATG，TrkA_R:GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR FRAG换货长度:400个碱基对（BP）。 Bax蛋白:IN5R:GTT GAC CAG AGT GGC GTA GG，Ex5F:TGA TCA GAA CCA TCA TG，NeoR:CCG CTT CCA TTG CTC AGC GG; PCR片段长度:野生型，304基点; Bax的空，507基点。 TauLacZ:lacZup:ACA ACG TCG TGA GTG GGA AAA，lacZdown:ATC AAC ATT AAA TGT GAG CGA G组; PCR片段长度:360基点。 </li><li>运行的PCR与TrkA的下列程序:步骤1:1分钟95℃，第2步:10秒，在95℃，第3步:20秒68℃，第4步:30秒72℃。 40次4 - 重复步骤2。 <ol><li>为的LacZ和Bax，运行的PCR使用以下程序:步骤1:在95℃下1分钟，步骤2:10秒，在95℃，第3步:1分钟在54℃，步骤4:1，在72分钟°C，重复步骤2 - 4 35个循环。 </li></ol></li><li>上，在100 V在1×Tris-醋酸盐-EDTA缓冲液的DNA梯的泳道2％琼脂糖凝胶进行20分钟运行的PCR样品，以评估片段长度。 </li><li>对于野生型的TrkA，T的存在分析胚胎RKA-tauLacZ和Bax无效等位基因。实验胚胎的基因型是TrkA的taulacZ / WT:Bax蛋白- / -和TrkA的taulacZ / taulacZ:Bax蛋白- / - ，取决于实验的问题。胚胎缺乏tauLacZ记者将负轴突染色，因此可以作为阴性对照来校准染色过程。 </li></ol> 3.胚胎染色<ol><li>使用商业套件的lacZ染色在3.2节下面介绍一些修改- 3.8。在这里，用微孔套件。 </li><li>沉浸在缓冲液A胚胎至少1小时，在室温下轻轻摇晃。 </li><li>直接浸入在缓冲液B的胚胎为至少15分钟，在室温下轻轻摇动。 </li><li>而胚胎是在缓冲液A / B中，在37℃下预暖组织中相应的解决方案。使用将5ml每胚胎组织中相应的解决方案。 </li><li>当胚胎在缓冲液A / B，准备新鲜的5-BRomo- 4-氯-3-吲哚基α-D-吡喃半乳糖苷（X-gal的）以40毫克/毫升在100％二甲基亚砜（DMSO）的浓度。 </li><li>稀X-gal的成以1mg / ml的浓度的预热组织基本溶液和再加5毫升X-gal的/组织中相应混合物到玻璃闪烁瓶中。 </li><li>转移的胚胎含有X-gal的/组织中相应混合物的玻璃闪烁管中。密封的盖子封口膜，37℃过夜，检查蓝染色存在。 </li><li> Postfix的胚胎与新鲜的4％PFA，pH值7.4。轻轻摇动4小时，4℃。 </li><li>在冷PBS漂洗胚胎两次。 </li></ol> 4.组织脱水及结算所<ol><li>为30分钟，在室温下，摇动在玻璃小瓶中发生的胚胎在50％甲醇/ 50％PBS中的混合物。 </li><li>继续在75％甲醇/ 25％PBS中脱水30分钟，在室温下摇动在玻璃小瓶中。 </li><li>继续在90％的脱水甲醇/ 10％的PBS 30分钟，在室温下摇动在玻璃小瓶中。 </li><li>继续在100％甲醇进行脱水30分钟（2次），在室温下摇动在玻璃小瓶中。 </li><li>而胚胎脱水，制备1:1（体积:体积）苯甲酰醇和苯甲酰苯甲酸的混合物。 注:苯甲酰醇和苯甲酰苯甲酸酯是有毒的。 </li><li>沉浸胚胎，在1:1（体积:体积）100％甲醇的混合物:100％BABB 15分钟。只使用玻璃，因为BABB会溶解塑料。 </li><li>胚胎沉浸在100％BABB彻底清除组织和可视化X-gal染色的清除胚胎。 注:尽快，因为X-gal的染色会褪色随着时间的推移图像。 </li></ol> 5.全山成像<ol><li>稳定的胚胎，沉浸在100％BABB，在适当的角度，使用摄影金属镊子。照亮使用的上行光并根据光的光源的组合。 </li><li>用解剖显微镜获取图像配备的数码相机。取多个明场曝光于五个连续焦平面相距0.5毫米沿Z轴。曝光时间和光圈值可以根据照明和摄像机的规格变化，并且需要为每个设置进行优化。 </li><li>使用标准的图像处理软件来处理的叠加图像，并生成合成照片。 </li></ol>

<ol>
	<li>Verze, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+innervation+in+hereditary+sensory+and+autonomic+neuropathy+type+IV.">Cutaneous innervation in hereditary sensory and autonomic neuropathy type IV.</a> Neurology. 55, 126-128 (2000).</li><li>Sethna, N. F., Meier, P. M., Zurakowski, D., Berde, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+sensory+abnormalities+in+children+and+adolescents+with+complex+regional+pain+syndromes.">Cutaneous sensory abnormalities in children and adolescents with complex regional pain syndromes.</a> Pain. 131, 153-161 (2007).</li><li>Uceyler, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+fibers+in+Fabry+disease:+baseline+and+follow-up+data+under+enzyme+replacement+therapy.">Small fibers in Fabry disease: baseline and follow-up data under enzyme replacement therapy.</a> J Peripher Nerv Syst. 16, 304-314 (2011).</li><li>Reichardt, L. F., Mobley, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+the+distance,+or+not,+with+neurotrophin+signals.">Going the distance, or not, with neurotrophin signals.</a> Cell. 118, 141-143 (2004).</li><li>White, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronous+onset+of+NGF+and+TrkA+survival+dependence+in+developing+dorsal+root+ganglia.">Synchronous onset of NGF and TrkA survival dependence in developing dorsal root ganglia.</a> J Neurosci. 16, 4662-4672 (1996).</li><li>Farinas, I., Wilkinson, G. A., Backus, C., Reichardt, L. F., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neurotrophin+and+Trk+receptor+functions+in+developing+sensory+ganglia:+direct+NT-3+activation+of+TrkB+neurons+in+vivo.">Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo.</a> Neuron. 21, 325-334 (1998).</li><li>Markus, A., Zhong, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+and+akt+mediate+distinct+aspects+of+sensory+axon+growth.">Raf and akt mediate distinct aspects of sensory axon growth.</a> Neuron. 35, 65-76 (2002).</li><li>Kuruvilla, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neurotrophin+signaling+cascade+coordinates+sympathetic+neuron+development+through+differential+control+of+TrkA+trafficking+and+retrograde+signaling.">A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling.</a> Cell. 118, 243-255 (2004).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raf+kinase+signaling+functions+in+sensory+neuron+differentiation+and+axon+growth+in+vivo.">Raf kinase signaling functions in sensory neuron differentiation and axon growth in vivo.</a> Nature. 10, 598-607 (2007).</li><li>Bulfone, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+olfactory+sensory+map+develops+in+the+absence+of+normal+projection+neurons+or+GABAergic+interneurons.">An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons.</a> Neuron. 21, 1273-1282 (1998).</li><li>Moqrich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressing+TrkC+from+the+TrkA+locus+causes+a+subset+of+dorsal+root+ganglia+neurons+to+switch+fate.">Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.</a> Nature. 7, 812-818 (2004).</li><li>Knudson, C. M., Tung, K. S., Tourtellotte, W. G., Brown, G. A., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax-deficient+mice+with+lymphoid+hyperplasia+and+male+germ+cell+death.">Bax-deficient mice with lymphoid hyperplasia and male germ cell death.</a> Science. 270 (5233), 96-99 (1995).</li><li>Lentz, S. I., Knudson, C. M., Korsmeyer, S. J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+support+the+development+of+diverse+sensory+axon+morphologies.">Neurotrophins support the development of diverse sensory axon morphologies.</a> J. Neurosci. 19, 1038-1048 (1999).</li><li>Patel, T. D., Jackman, A., Rice, F. L., Kucera, J., Snider, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sensory+neurons+in+the+absence+of+NGF/TrkA+signaling+in+vivo.">Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.</a> Neuron. 25, 345-357 (2000).</li><li>Donovan, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-RAF+kinase+drives+developmental+axon+growth+and+promotes+axon+regeneration+in+the+injured+mature+CNS.">B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS.</a> The Journal of experimental medicine. 211, 801-814 (2014).</li><li>Mercer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+endogenous+oncogenic+V600EB-raf+induces+proliferation+and+developmental+defects+in+mice+and+transformation+of+primary+fibroblasts.">Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts.</a> Cancer research. 65, 11493-11500 (2005).</li><li>Tronche, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+glucocorticoid+receptor+gene+in+the+nervous+system+results+in+reduced+anxiety.">Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety.</a> Nature genetics. 23, 99-103 (1999).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Neuronal+Subsets+in+Transgenic+Mice+Expressing+Multiple+Spectral+Variants+of+GFP.">Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Schmidt, H., Rathjen, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI-labeling+of+DRG+neurons+to+study+axonal+branching+in+a+whole+mount+preparation+of+mouse+embryonic+spinal+cord.">DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord.</a> J Vis Exp. , (2011).</li></ol>

The authors declare that there are no competing interests.

胚胎的TrkA taulacZ小鼠的上述X-gal染色过程允许长距离轴突突起，在完好的固定胚胎的快速和详细可视化。因为Bax缺无背景这些小鼠允许信令可能有助于既轴突生长和神经元存活机制的探测。有兴趣的转基因或基因敲除小鼠交配允许轴突表型的综合评估，可以服务于未来的实验作为有用的指导，研究轴突生长的信号更详细的方式。在体内轴突生长研究的一个主要挑战是准备组织一次段和评估生长轴突可能受影响的多种方式通过任何一项遗传操作的复杂的表型。该TrkA的taulacZ / taulacZ:Bax蛋白- / -行使一个基因的全面影响评估对周围伤害感受器轴突发展的兴趣。 该技术是仅限于伤害感受器轴突轨迹，在早期发育阶段通常会表达TrkA的可视化。除了外周伤害感受器，它可能显现交感神经和基底前脑胆碱能预测;然而这将需要的染色技术特定优化组织清算是这里所描述的协议的关键一步。它能够在全安装的准备更深躺在预测，因而不同的表型的潜在鉴定详细评估。荧光报告，如TdTomato或GFP的记者18,19，可以提供在特定条件下的视觉清晰度，然而，很难获得整个贴装图像，因为我们已经发现，在相对 ​​于在X-gal染色，荧光信号期间淡出基本上组织清算程序。 DiI标记染色也被广泛用于轴突标记20;其缺点是，它不能选择性地标记轴突的特定亚型，如伤害感受器纤维这里，并且难以在周边以充分标记长轨迹。 总之，我们提供了一个使用两个转基因小鼠系的组合，以允许在小鼠胚胎痛觉神经轴突的充分乔木的常规可视化的协议。

建立精确的神经网络是一个复杂的发育过程中神经系统的功能是必不可少的。干扰在这个过程导致神经元功能障碍已与人类神经系统疾病1-3。研究轴突生长和靶神经支配的哺乳动物的分子机制，我们开发了一个协议，以可视化的TrkA表达感觉神经元使用两种转基因小鼠系的组合的轴突轨迹。 TrkA的是受体对神经生长因子NGF和是伤害性感觉神经元4的功能标记。早期发育过程中的TrkA高度表达在伤害感受神经元，并介导NGF依赖性神经元存活，轴突生长，树枝状和靶神经支配5-9。在的TrkA taulacZ小鼠中，野生型的TrkA基因置换为taulacZ表达式c磁带式10中 ，以使得推定的TrkA阳性神经元的轴突形态可通过β半乳糖苷酶（X-gal的）染色11可视化。使用杂的TrkA taulacZ / WT线，我们可以检查可调节或干扰体内感觉传入预测的发展的因素。 此外，TrkA的表达是不存在于纯合的TrkA taulacZ / taulacZ小鼠，这因此可以用来评估轴突生长促进机制在不存在NGF / TrkA的信令。由于疼痛神经元依赖于NGF / TrkA的信号不仅对轴突的生长，同时也为求生存，我们另外聘请鼠标线，缺乏促凋亡蛋白Bax基因，抑制细胞凋亡在胚胎DRG神经元细胞死亡是另有解救出来在不存在的TrkA信令的观察。的Bax蛋白- / -背景12从而允许molecu信号通路专门影响轴突生长7-9,13-15的LAR解剖。在的TrkA - / - :Bax蛋白- / -小鼠，DRG神经元存活，但在皮肤感觉传入神经 ​​支配被完全取消14,15。我们可以有选择地激活的信号通路，以确定它们各自的轴突预测的发展作出了贡献。该方法的效用是，它允许改变在轴突生长表型的评估当不同的遗传修饰的繁殖到的TrkA taulacZ / taulacZ:Bax蛋白- / -或TrkA的taulacZ / WT:Bax蛋白- / -背景。

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

全长神经元的预测在胚胎的可视化是必须获得的神经网络如何发展哺乳动物的认识。在这里，我们描述的方法原位标记背根神经节（DRG）轴突预测的一个子集，使用多个基因操纵鼠标线，以评估他们的表型特征。所述的TrkA阳性神经元是伤害性感受器神经元，专门用于疼痛信号的传输。我们利用的TrkA taulacZ鼠标线标记所有的TrkA阳性轴突末梢的轨迹在完整的小鼠胚胎。我们进一步繁殖的TrkA taulacZ线到Bax缺无的背景，这基本上废除神经细胞凋亡，以评估的独立地对神经元存活的遗传操作的可能影响生长相关的问题。随后，感兴趣的转基因小鼠繁殖与TrkA的taulACZ / Bax的零线和然后准备用于使用本文所描述的技术的研究。此报告包括解剖，组织准备，染色和结算的时间对小鼠繁殖计划的详细信息，基因分型，以便在整个安装准备全长轴突轨迹可视化。

TrkA的WT / taulacZ的基因型:Bax蛋白- / -和TrkA的taulacZ / taulacZ:Bax蛋白- / -胚胎可以通过标准PCR基因分型来明确地确定（ 图1）。 X-gal染色显示在常规染色的胚胎（ 图2，图3a）详述周轴突乔木皮下，并在整个组织清除后的胚胎（ 图3b，4）。 我们已经孕育了TrkA的WT / taulacZ:Bax蛋白- / -与下神经元特异性巢子17控制携带组成激酶激活B-RAF（V600E）突变（LSL-kaBraf 16）自驾路线，获得LSL- kaBraf:TrkA的taulacZ / taulacZ:Bax蛋白- / -小鼠。在这些胚胎，这完全缺乏TrkA的信令，伤害感受器圆周突起的形态仍然开发了近正常（ 图4）。这证明对B-RAF的信令在外设轴突生长，并且还SA关键功能显示，存活和轴突生长促进的TrkA信令的功能可以被分离出来，与Bax蛋白- / -遗传背景代替的TrkA的依赖性存活信号的损失。 <img alt="图1" src="/files/ftp_upload/52212/52212fig1highres.jpg" /> 图1:转基因小鼠的基因分型代表琼脂糖凝胶图像。示例1:TrkA的WT / taulacZ:Bax的WT / - ，样品2:TrkA的taulacZ / taulacZ:Bax蛋白- / - ，样品3:TrkA的WT / taulacZ:Bax蛋白- / - 。 <img alt="图2" src="/files/ftp_upload/52212/52212fig2highres.jpg" /> 图2:E18.5的TrkA WT / taulacZ小鼠染色用X-gal从三叉神经节轴突突起在躯干和HEA。d的显示在一个未清除的胚胎。比例尺:1毫米。 <img alt="图3" src="/files/ftp_upload/52212/52212fig3highres.jpg" /> 图3:前E18.5 TrkA的WT / taulacZ鼠标染色用X-gal（A）及（B）与BABB从病种付费中旬躯干预测结算后显示。比例尺:2毫米。 <img alt="图4" src="/files/ftp_upload/52212/52212fig4highres.jpg" /> 图4:一个E16.5 LSL-kaBraf:TrkA的taulacZ / taulacZ:Bax蛋白- / - :巢蛋白的Cre小鼠胚胎染色用X-gal和清除与BABB kaBraf编码激酶活化的B-Raf激酶蛋白B-RAF V600E。 KAB-RAF的DRG神经元中的表达导致了全长，接近正常的轴突延长在没有TrkA的信令。比例尺:1毫米。欲了解更多的图像，包括控制，见文献。 15， 图2。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td colspan="2">用于PCR的基因分型的引物</td></tr><tr><td rowspan="3"> 1 </td><td> TrkA的</td><td> DNA片段大小:400基点</td></tr><tr><td> TrkA_F </td><td> GCG GGC GCC GCC GCG ATG </td></tr><tr><td> TrkA_R </td><td> GAA GCC GCC TGC GCG GCT CTG C​​CA GGG TG </td></tr><tr><td rowspan="4"> 2 </td><td> Bax基因</td><td> DNA片段大小:304 bp的（野生型），507基点（BAX空） </td></tr><tr><td> IN5R </td><td> GTT GAC CAG AGT GGC GTA GG </td></tr><tr><td> Ex5F </td><td> TGA TCA GAA CCA TCA TG </td></tr><tr><td> NeoR </td><td> CCG CTT CCA TTG CTC AGC GG </td></tr><tr><td rowspan="3"> 3 </td><td> TauLacZ </td><td> DNA片段大小:360基点</td></tr><tr><td> lacZup </td><td> ACA ACG TCG TGA GTG GGA AAA </td></tr><tr><td> lacZdown </td><td> ATC AAC ATT AAA TGT GAG CGA摹</td></TR&gt; </tbody></table> 表1:用于PCR的基因分型的引物。

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.0K 次观看.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

视频: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

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 ...

科研

神经科学

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

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 ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

小鼠皮肤及其毛囊图案和感觉轴突形态分析中的应用平山成像

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

成像钙<sup&gt; 2+</sup&gt;在小鼠视网膜锥光感受器轴突终末动态

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...