我们感谢他的帮助下，尤里M · Usachev博士/鼠标足底冲床活检免疫共聚焦显微镜成像的初步标准化;和唐娜L。哈蒙德博士为她继续在这项工作中的帮助和建设性的批评。这项工作是由从NINDS / NIH（NS069898）的赠款，并从国防部前列腺癌研究计划署（DOD - PCRP 101096）的一个想法发展到DPM的补助金

1。动物灌注在这项研究中的所有动物进行的程序是机构动物护理和使用爱荷华大学委员会批准，并按照NIH的指导方针，在研究中使用动物。 <ol><li>在灌注前一天，准备1 L的磷酸盐缓冲液（0.2 M PB，pH值7.4双蒸H 2 O），并储存在4 ° C这将是用于灌注和后固定过程。 </li><li>灌注一天，准备500毫升的4.0％多聚甲醛在0.1M PB（煤灰，pH值7.4）固定液，音量足够灌注2小鼠：微波200毫升的 DDH 2 O在玻璃烧杯，持续30秒或直到它接近沸点。与通风柜下不断搅拌烧杯中添加20克颗粒多聚甲醛（PFA）。加入5毫升的5 N NaOH下降明智和搅拌，直到溶液清除。当煤灰完全溶解，冷却至室温（RT）的解决方案。和Slowly加入250毫升的0.2 M PB，同时不断搅拌。使用逐步较弱盐酸解决方案（6 N到1 N到0.1 N的），调整pH值至7.4。调整最终体积至500毫升和冰浴。还准备从0.2 M的股票在DDH 2 O和寒意冰1:1稀释400毫升为0.1米PB 。 </li><li>麻醉intraperitonial注射过量的麻醉（戊巴比妥钠，80毫克/公斤，与27G针管理）鼠标。 </li><li>在灌注，50毫升0.1M的PB（稀释0.2 M PB股票1:1与DDH 2 O）和150毫升煤灰顺序被传递到老鼠的流通。蠕动泵，填补用PB管，一端固定在一个23 1 / 2 G蝴蝶针。油管的另一端浸入含有0.1中号PB的烧杯。通过设置足够的金额，以确保有管路中无气泡的一个解决方案的速度10毫升/分钟的蠕动泵和泵。 </li><li>等待，直到在一个nesthetized鼠标不再显示任何反射活动，如脚趾/尾捏和眨眼反射。奠定一个夹层托盘腹侧通风柜内的动物和安全的爪子，用胶带。喷雾用70％乙醇的皮毛。通过皮肤，沿中线切口，显露胸腔和腹壁至上季度。然后，穿过腹壁，胸腔的基础。此切口后，膈肌和较低的胸骨年底应可见。通过膜片的保证金左到右，沿胸骨全长小心剪去，注意不要损害肺部。从胸骨在这个阶段的一些轻微出血是正常的，不会妥协的灌注。一旦切口沿胸骨已经取得了膜片切割，它应该是可以解除的肋骨向上和向外，这样暴露心脏是各占一半。移动预留的肺部如果有必要。胸腔的两个半在这个位置上使用止血夹子举行。插入的蝴蝶针（23 1 / 2 G）的蠕动泵进入左心室3-4毫米，动物的长轴平行。这安置最大限度地减少了针脱落的风险。做一个小切口右心房允许返回循环流动的心脏。 </li><li>用0.1 M PB在开始灌注10毫升/分钟4-5分钟。如果仍然有显著的右心房，在这一点产生的血液量，继续，直到流是明确的。 </li><li>停止PB的流量和交换机上蠕动泵入口到4％PFA解决方案。降低流速至5毫升/分钟和20-25分钟，恢复抽水煤灰解决方案。由于煤灰进入流通，肌肉痉挛，几分钟后，应当在动物字面意思是“固定”的位置。这种刚性可以轻轻推对hindpaws，照顾不搬动物测试ND打跑套管。良好灌注，防止肢体弯曲响应。一旦与煤灰灌注完成，插管和止血夹子可以断开连接，并准备组织剥离的尸体。夹层采用的方法取决于具体组织。 </li><li>准备4％PFA / 5％（V / V）苦味酸（PA;收集和固定后仅足底冲床）加入饱和苦味酸溶液，4％的煤灰。列入PA的显著提高抗原1在周围神经组织的可探测性</li><li>准备骨/软骨组织脱钙/冷冻保护剂的解决方案 - 0.1M的PB 10％EDTA，0.07％甘油和15％的蔗糖。 EDTA为基础的解决方案，同时保留表位 2除垢组织。 </li><li>准备的冷冻保护剂的解决方案 - 30％在0.1 M PB蔗糖。 </li></ol> 2。组织剥离/去除，后固定和切片<ol><li> 足底冲床 放置的灌注SED动物腹侧切割垫或其他坚固的表面上。抱足跖面，按打孔活检工具（3毫米直径;哈里斯微冲，特德佩拉公司）牢固地插入脚的中间。通过180度转动活检工具来回确认通过后爪全部切活检工具。从脚下轻轻地取出活检工具和退出到第2毫升无菌管组织，含有1毫升4％/ 5％煤灰PA解决方案。 </li><li> 肢体骨（如股骨） 沿骨盆水平的动物的回一个外侧切口，两侧后肢持续下降。切成骨盆周围的肌肉分开股骨，骨盆，而离开股骨近端头完好无损。胫/腓骨切离开远端头完好无损，消除骨干周围肌肉/骨膜。股骨放入2毫升中含1毫升4％PFA /管5％的PA解决方案。 </li><li>修复后在4％/ 5％的煤灰PA解决方案的组织样本，轻轻混合16-18 h摇杆，在4 ° C </li><li>除垢的组织如下： 足底冲床 （除垢脚下的小骨头）地方与第2毫升在无菌管组织冲床，包含在0.1 M PB 1.5与0.07％甘油和15毫升10％EDTA溶液％的蔗糖。放在摇杆与轻度混合为16-18 h，在4 ° C。 对于四肢骨骼的地方组织与第2毫升在无菌管，10％EDTA溶液1.5毫升含0.07％的甘油在0.1 M PB与管15％的蔗糖。与轻度混合摇杆放在管6-7天，在4 ° C脱钙液应改为每24小时和一双镊子刚性损失监察组织。 </li><li>转移到无菌的2 ml管组织第1.5毫升的冷冻保护剂溶液（30％在0.1 M PB蔗糖），放在摇杆瓦特管第i个轻度混合16-18小时于4 ° C。 </li><li>准备组织切片的最佳切削温度的床体积（OCT）的化合物在低温恒温器安装组织块（樱花Finetek美国公司）和允许冻结在低温恒温器室（通常维持在-20 ° C）。 A低温气溶胶（细胞冻结，美国控制有限公司），也可以喷洒在华侨城加速冻结过程。放在床的华侨城组织标本，并与华侨城一层薄薄的额外覆盖。轻轻喷低温气溶胶华侨城，直到它已硬化和组织内冻结。允许将试样在低温恒温室到切割头和组织标本块切削温度平衡 - 至少1小时。试图以节时嵌入化合物仍是太冷结果脆性部分和组织损伤。 </li><li>一旦组织已经达到了最佳的切削温度，开始切割试样，产生40微米的部分。 </li><l我收集到12孔组织培养板10毫米的叠氮化钠含有0.1 M的PB&gt;足底冲床的冰冻切片。确保部分仍然淹没在此解决方案，在4 ° C节可以存储在这种方式为4-6个月免疫目的。另外，自由浮动的部分，可以无限期地储存在-20 ° C（500毫升0.1 M PBS，pH值7.2，蔗糖30克，10克，300毫升乙二醇PVP40）的冷冻保护剂溶液。用蒸馏水调整终体积至1L）3。肢骨收集的冰冻切片直接到明胶预涂幻灯片（或Superfrost 加幻灯片，尔科技），让风干1小时，然后储存在​​-20 ° C在这种方式存储的幻灯片上的骨部分可用于2-3个月内免疫目的。 </li></ol> 3。感觉神经纤维的免疫染色组织切片 3.1。足底冲床部分 - 浮动部分染色<ol><li>使用刀片，切6-7毫米，从1毫升的微量提示。在0.1M的PB多次吸1％胎牛血清（FBS），（抑制坚持组织切片提示墙壁）的尖端内部前置条件。足底一拳部分转移到24孔组织培养板染色。一般8-10部分应染色，每口井，以确保高品质的几个部分，每免疫产生。 </li><li>洗净用500μl0.1 M的PB每口井为大力在RT摇杆搅拌5分钟的足底冲床部分。重复2次以上。 </li><li>倒掉洗涤液，封闭液，10％山羊血清和0.3在0.1％的Triton X - 100米PB（500μL封闭液，每孔）组成孵化足底冲床部分轻轻混合为1小时摇杆，在4 ° C </li><li>丢弃阻塞的解决方案，并组织切片孵育在小学antiboDY /抗体稀释温和混合摇杆为18-24 h，在250μL封闭液在4 ° C根据研究者的实验要求，单一的免疫标记（对一个特定的蛋白质或神经纤维标记抗体），或双免疫标记（对两种蛋白质或神经纤维标志的主要有两个抗体）可以在同一节。在执行双重免疫标记是很重要的，以验证使用的两个主要抗体是提出在不同的宿主物种（例如，一个在小鼠和兔提出提出），或者，纯化不同的免疫球蛋白G（IgG）的亚型（如单克隆抗体IgG1和IgG2a）都可以使用。可取的做法是，用封口膜密封板一夜之间孵化，以避免水分蒸发过快。为了周边感觉神经纤维染色，一些特异性抗体可以使用。对蛋白抗体神经丝蛋白200（NF200;西格玛）标签LARGE -直径的纤维，而降钙素基因相关肽（CGRP;西格玛）和瞬时受体电位香草1（TRPV1受体，Neuromics）标签肽，小直径的外周感觉神经纤维。所有末梢神经纤维也可与β3-微管蛋白抗体染色。皮肤，IV型胶原（Abcam公司）是用来描述分离的表皮从真皮的基底膜。作为一般规则，抗体需要培养的细胞为基础的免疫检测，通常在5-10微克/毫升的浓度，超过集中的5-10倍。 </li><li>组织切片洗净与500μL封闭液（TRITON X - 100）每5分钟的剧烈混合在RT摇杆。重复3次以上。 </li><li>丢弃在适当的荧光标记的二抗温和混合摇杆（通常稀释1:1000封闭液，500μL）的清洗液和孵化组织切片在4 ° C。板块亩被包裹在铝箔，以避免光漂白的荧光团。 </li><li>洗净阻塞的解决方案，每以及10分钟的剧烈混合在RT摇杆与500μL组织切片。大力为在室温下10分钟搅拌，然后用500μL0.1 M的PB。最后，用500μL0.05 M PB大力为在室温下10分钟搅拌。 </li><li> FBS的治疗头（从末端切6-7毫米）使用1毫升微量的，吸的部分，从组织培养板和转移到SuperFrost 加上显微镜玻片上。安排的部分，并用细的画笔吸收多余的洗涤缓冲液。避免长时间暴露在光线下。在室温下孵育的幻灯片，为了让部分干上滑动的部分（5-30分钟）。 </li><li>申请安装介质（ProLongGold，Vectashield或类似）数滴，慢慢地放到部分玻璃盖玻片。允许的幻灯片，在黑暗中坐5分钟，然后密封盖玻片边缘透明的指甲波兰。允许空气干燥15-20分钟。幻灯片现在可以没有任何显著的荧光损失几年的可视化或储存在-20 ° C。 </li></ol> 3.2。肢体骨部分 - 幻灯片染色<ol><li>允许的幻灯片返回到RT从-20 ° C储存。使用疏水性障碍笔（超巴氏液体阻塞钢笔，特德佩拉公司）circumscribe该地区的含骨与解决方案的慷慨层染色切片的幻灯片。允许空气干燥10-15分钟。 </li><li>洗骨部分与250μL0.1 M的PB每张幻灯片为5分钟，重复2次以上。 </li><li>弃洗涤液，封闭液，10％的山羊血清和0.3％的Triton X - 100的在0.1中号PB阻止每张幻灯片的解决方案（250μL）1小时4 ° C。组成孵化骨节</li><li>丢弃阻塞的解决方案，并在主抗体（或初级抗体结合孵育骨部分，在案件根据3.1.4中提到的双重免疫标记）稀释250μL封闭液18-24 h，在4 ° C。这是非常重要的需要注意的幻灯片必须放置在一个密封的盖子湿盒，以避免初级抗体溶液干燥。为了周边感觉神经纤维染色，上述设置（见3.1.4节）的抗体，可以用类似的工作浓度。 </li><li>每250μL封闭液在室温下15分钟的幻灯片（TRITON X - 100）组织切片洗净，重复3次以上。 </li><li>弃洗涤液，骨部分在适当的共轭荧光抗体（通常稀释于封闭液，250μL1:1000）孵育4 ° C。染色室必须包裹在铝箔，以避免光漂白的荧光团。为方便起见，最好是使用深色的染色商会（如幻灯片孵化托盘/盒，RPI的公司）。 </li><li> W灰堵在室温为15分钟，每张幻灯片的解决方案与250μL的骨骼部分。在室温为15分钟，然后用500μL0.1 M的PB。最后，用500μL0.05 M PB在室温下15分钟。浸在DDH 2 O简要冲洗幻灯片，然后在室温下孵育，为了让骨部分空气干燥（5-30分钟） 。避免长时间暴露在光线下。 </li><li>申请安装介质（ProLongGold或类似）数滴，慢慢地放到部分玻璃盖玻片。让幻灯片站在黑暗5分钟，然后用透明的指甲油密封盖玻片的边缘。允许干燥15-20分钟。幻灯片可存放数年无荧光的损失，在-20 ° C。 </li></ol> 4。代表性的成果 4.1。足底冲床组别足底冲床组织切片可以epifluorescence显微镜下，或共聚焦显微镜下一个10X，40X或63X的目标。 <img alt="图1" src="/files/ftp_upload/3485/3485fig1.jpg" /> 图1。广告显示基底膜IV型胶原抗体（绿色）染色，在表皮真皮交界处的软骨和肌肉，。许多CGRP -（AB，红色）和NF200阳性纤维（CD，红）是分布在整个足底地区的老鼠的皮肤（箭头）。 β3-微管蛋白是泛神经元标记（E，红色），而辣椒素染色（红色）主要限于小直径的纤维也CGRP的阳性（绿色;箭头）。 A和C是10倍的目标（比例尺-500微米）的epifluorescence图像，B，D，E和F下采取的一个60X的目标（规模11影像在2微米的增量Z - Stack的共聚焦图像生成的复合材料酒吧 - 50微米）。 4.2。四肢骨节肢体骨组织切片也可以可视化epifluorescence显微镜下，或下焦microscopE一个10X，40X或63X的目标。 <img alt="图2" src="/files/ftp_upload/3485/3485fig2.jpg" /> 图2显示了免疫与抗- CGRP（AB，红色;箭头），抗- NF200（CD，红色;箭头），抗β3-微管蛋白（E，红色）和反辣椒素（F，红）合作染抗- CGRP的抗体（绿色箭头）。这些图像显示的感觉神经纤维分布在小鼠股骨的海绵头地区的骨基质的亚型。 A和C是10倍的目标（比例尺-500微米）的epifluorescence图像，B，D，E和F下采取的一个60X的目标（规模11影像在2微米的增量Z - Stack的共聚焦图像生成的复合材料酒吧 - 50微米）。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraepidermal+nerve+fiber+loss+corresponds+to+the+development+of+Taxol-induced+hyperalgesia+and+can+be+prevented+by+treatment+with+minocycline.">Intraepidermal nerve fiber loss corresponds to the development of Taxol-induced hyperalgesia and can be prevented by treatment with minocycline.</a> Pain. 152, 308-313 (2011).</li><li>Bloom, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer-Induced+Bone+Remodeling,+Skeletal+Pain,+and+Sprouting+of+Sensory+Nerve+Fibers.">Breast Cancer-Induced Bone Remodeling, Skeletal Pain, and Sprouting of Sensory Nerve Fibers.</a> The Journal of Pain. 12, 698-711 (2011).</li><li>Constantin, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+Tumor+Necrosis+Factor+&#945;+(TNF&#945;)+Requires+TNF+Receptor+Type+2+to+Generate+Heat+Hyperalgesia+in+a+Mouse+Cancer+Model.">Endogenous Tumor Necrosis Factor &#945; (TNF&#945;) Requires TNF Receptor Type 2 to Generate Heat Hyperalgesia in a Mouse Cancer Model.</a> J. Neurosci. 28, 5072-5081 (2008).</li><li>Jankowski, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitization+of+Cutaneous+Nociceptors+after+Nerve+Transection+and+Regeneration:+Possible+Role+of+Target-Derived+Neurotrophic+Factor+Signaling.">Sensitization of Cutaneous Nociceptors after Nerve Transection and Regeneration: Possible Role of Target-Derived Neurotrophic Factor Signaling.</a> The Journal of Neuroscience. 29, 1636-1647 (2009).</li><li>Jimenez-Andrade, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+Sprouting+of+Adult+Nociceptors+in+Chronic+Prostate+Cancer-Induced+Bone+Pain.">Pathological Sprouting of Adult Nociceptors in Chronic Prostate Cancer-Induced Bone Pain.</a> J. Neurosci. 30, 14649-14656 (2010).</li><li>Persson, A. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium-calcium+exchanger+and+multiple+sodium+channel+isoforms+in+intra-epidermal+nerve+terminals.">Sodium-calcium exchanger and multiple sodium channel isoforms in intra-epidermal nerve terminals.</a> Molecular Pain. 6, 84-84 (2010).</li><li>Ohshima, M., Miyake, M., Takeda, M., Kamijima, M., Sakamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+Enterotoxin+B+Causes+Proliferation+of+Sensory+C-Fibers+and+Subsequent+Enhancement+of+Neurogenic+Inflammation+in+Rat+Skin.">Staphylococcal Enterotoxin B Causes Proliferation of Sensory C-Fibers and Subsequent Enhancement of Neurogenic Inflammation in Rat Skin.</a> Journal of Infectious Diseases. 203, 862-869 (2011).</li><li>Johnson, M. S., Ryals, J. M., Wright, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+loss+of+peptidergic+intraepidermal+nerve+fibers+in+an+STZ-induced+mouse+model+of+insensate+diabetic+neuropathy.">Early loss of peptidergic intraepidermal nerve fibers in an STZ-induced mouse model of insensate diabetic neuropathy.</a> Pain. 140, 35-47 (2008).</li></ol>

没有利益冲突的声明。

在这里，我们有详细的老鼠的皮肤和外周感觉神经纤维的免疫组化和检测骨组织切片的制备方法。从足底打孔活检产生的部分包含皮肤光洁，毛，这意味着可用于任何皮肤类型的协议。这些技术也可以在这些组织染色的其他类型的细胞（如白细胞，血管内皮细胞，平滑肌等等）。这些方法提供之间最佳的超微结构保存很好的折衷（这是由戊二醛固定实现，但经常在表位的破坏和削弱免疫组化染色?...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录号 </td><td> 评论 </td></tr><tr><td> 3毫米哈里斯微冲</td><td>材料</td><td>泰德佩拉</td><td> 15094 </td><td></td></tr><tr><td>灌注泵</td><td>材料</td><td> VWR International的</td><td> 23609-170 </td><td></td></tr><tr><td>多聚甲醛</td><td>试剂</td><td> Fisher Scientific则</td><td> T353 </td><td></td></tr><tr><td>苦味酸</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> 239801 </td><td></td></tr><tr><td>华侨城嵌入化合物</td><td>试剂</td><td>组织Tek的</td><td> 4583 </td><td></td></tr><tr><td>细胞冷冻低温喷雾</td><td>材料</td><td>控制公司</td><td> 3118 </td><td></td></tr><tr><td>山羊血清</td><td>试剂</td><td> Sigma - Aldrich公司</td><td> G9023 </td><td></td></tr><tr><td>孵化托盘和免疫染色盖（大） </td><td>材料</td><td> RPI的公司</td><td> 248270（托盘） 248270 - A（盖） </td><td></td></tr><tr><td> ImmEdge疏水屏障笔</td><td>材料</td><td>媒介实验室</td><td> H - 4000 </td><td></td></tr><tr><td>骆驼毛的刷子（＃厚度1） </td><td>材料</td><td>泰德佩拉</td><td> 11859 </td><td></td></tr><tr><td>临长的金安装介质</td><td>试剂</td><td> Invitrogen公司</td><td> P36930 </td><td></td></tr></tbody></table>

tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

检测和外周感觉神经纤维的物理，化学和热感官刺激的初级加工的关键是在动物和人类的感官知觉。这些外设的感觉神经纤维表达过多的受体和离子通道蛋白的检测，并启动具体的感官刺激。方法可用来描述末梢感觉神经支配的皮肤纤维，它也可以用来识别特定的离子通道蛋白在这些纤维的功能表达的电气性能。然而，类似的电生理方法是不可用的（和支配其他内脏器官，包括最具挑战性的组织，如骨的外周感觉神经纤维受体和离子通道蛋白的功能表达的检测也难以发展）。此外，这种电生理方法不能被用来确定非兴奋性蛋白的表达S的外周感觉神经纤维。因此，周边感觉神经fivers /内脏组织样本的免疫提供尽可能最好的方式，以确定这些神经纤维的利益的特定蛋白质的表达。到目前为止，大多数在感觉神经元蛋白表达的研究利用免疫程序在感觉神经节，其中的信息是有限的特定类型或感觉神经元亚群的细胞体在特定的蛋白质表达。在这里，我们报告的编制外周感觉神经纤维免疫外设/内脏组织样本的详细方法/协议。我们编制的皮肤或足底打孔活检和骨（股骨）从小鼠免疫外周感觉神经纤维的部分具体细节方法。这些方法不仅末梢感觉神经元蛋白表达的定性测定的关键，但也提供了定量分析方法确定特定类型或亚群的感觉纤维蛋白表达水平的变化，以及确定在感觉神经纤维的数量和密度在各种病理状态的形态和/或解剖变化。此外，这些方法并不局限于只感觉神经纤维的染色，但也可以使用任何类型的神经纤维染色的皮肤，骨骼和其他内脏。

Watch this Scientific Journal Video about Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones at JoVE.com

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

末梢感觉神经纤维亚型的免疫细胞化学鉴定（和蛋白表达其中的检测）底层外围感觉的分子机制的理解是关键。在这里，我们描述外设/内脏组织样本，特定的免疫外周感觉神经纤维，如皮肤和四肢骨骼的制备方法。

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

tissue-preparation-immunostaining-mouse-sensory-nerve-fibers

Research

JoVE Journal

Neuroscience

23.0K 次观看.  The University of Iowa. 末梢感觉神经纤维亚型的免疫细胞化学鉴定（和蛋白表达其中的检测）底层外围感觉的分子机制的理解是关键。在这里，我们描述外设/内脏组织样本，特定的免疫外周感觉神经纤维，如皮肤和四肢骨骼的制备方法。

视频: Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

制备小鼠脑组织中的免疫电镜

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

科研

神经科学

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

成像遥感信息素在小鼠犁鼻急性组织切片的制备

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

一个孤立的半完整的上下游鼠标前庭上皮细胞电生理和高分辨率的双光子显微镜

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

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 Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

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 ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

人 Intraepidermal 神经纤维内线粒体三维成像及分析

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.

一种方法为Target和隔离气道支配感觉神经元的小鼠

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

从美国牛蛙的小囊（毛细胞的生理准备<em&gt;牛蛙</em&gt;）

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

嗅球的四次免疫染色用于嗅觉感觉轴突分子识别码的可视化

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...