Name: whole-mount confocal microscopy for adult ear skin: a model system to study neuro-vascular branching morphogenesis and immune cell distribution
Uploaded: 2018-03-29T15:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution at JoVE.com

我们感谢吉尔的实验室管理和技术支持, j. 霍金斯和国家卫生研究院 (NIH) 的工作人员50建立了援助老鼠护理的动物设施, 以及里德和 f. Baldrey 的行政援助。还感谢 s Motegi 和 m. Udey 分享他们的耳朵皮肤解剖协议, n. 烧伤的编辑帮助, 以及干细胞和神经血管生物学实验室成员的技术帮助和深思熟虑的讨论。KAITOKU 是日本促进科学协会 (jsp) 的支持。这项工作得到了国家心脏、肺和血液研究所 (HL005702-11 到 Y.M.) 的校内研究计划的支持。

这一节的所有实验都是在国家心脏、肺和血液研究所 (NHLBI) 动物护理和使用委员会的批准下进行的。
1. 成人小鼠耳皮收集
<ol><li>弄死成年小鼠的二氧化碳 (CO2) 暴露在一个封闭的房间, 然后确认安乐死的颈椎脱位。 注: 实验遵循国家卫生研究院 (NIH) 的安乐死方法指南。</li>
	<li>从底座解剖耳朵, 将其放置在 35 x 10 毫米的2培养皿中, 其中包含有2毫升的汉克平衡盐溶液 (HBSS)。用剪刀简单地修剪头发。</li>
	<li>从介入软骨中仔细剥离后皮肤和前皮肤。 注: 软骨附着于前皮肤。</li>
	<li>将后、前皮肤分别转移至24井板, 含1毫升冰鲜4% 多聚甲醛 (粉煤灰) 的磷酸盐缓冲盐水 (PBS) 每井。将4% 的粉煤灰中的后、前皮肤压扁。</li>
	<li>修复后和前皮肤与软骨在4°c 1 小时。</li>
	<li>在室温下, 在混合器上用温和的混合, 在1毫升的 PBS 中清洗后、前皮肤3次5分钟。</li>
	<li>将后、前皮肤与软骨转移到 35 x 10 毫米2培养皿的底部。切断基底区域, 折叠并有脂肪和结缔组织。用细弯钳剥离前皮肤软骨。</li>
	<li>用细弯镊子小心去除后皮肤内侧的毛发、脂肪组织和结缔组织。用 PBS 把皮肤弄湿。</li>
</ol>2. 小鼠耳皮肤的全层免疫组化染色
注: 以下各节中的所有实验均按照 NIH 实验室安全指南进行。
<ol><li>准备阻塞缓冲区。过滤器10% 热灭活山羊血清 (HIGS) 稀释在 pbs 与0.2% 海卫 X-100 (TX-100) 阻断缓冲, 或10% 驴血清 (DS) 稀释在 pbs 与 0.2% TX-100 阻塞缓冲区, 使用0.45 µm 过滤器单位。</li>
	<li>将后、前皮肤转移至24井板, 其中包含1毫升 10% HIGS 阻塞缓冲器或 10% DS 阻塞缓冲器。在室温下, 在搅拌机上用温和的混合孵育皮肤30分钟。</li>
	<li>在阻塞缓冲器 (10% HIGS 或 10% DS) 中稀释主要抗体 (材料表), 准备主要抗体溶液。 注: 全分队城市免疫组化分析成人耳皮肤抗体的泛神经元标记神经元特定类 III β-蛋白 (Tuj1, 兔多克隆 IgG 或小鼠 monocloncal IgG2a, 1:500 稀释在最后浓度的2µg/毫升),pan 内皮细胞标记的血小板内皮细胞黏附分子 1 (PECAM-1, 仓鼠单克隆 IgG, 1:300 稀释在最后浓度3.3 µg/毫升), 髓鞘标记髓鞘碱性蛋白 (MBP, 兔多克隆 IgG, 1:200 稀释在最后浓度的5µg/毫升) 和炎症髓细胞标记 CD11b (大鼠单克隆 IgG2b, 1:50 稀释在最后浓度20µg/毫升) 显示在图 1和图 2。以 Cy3-conjugated 抗体为血管平滑肌细胞标记α平滑肌肌动蛋白 (αSMA) 与继发抗体 (2.6) 一起孵化皮肤。我们自己测试的主要抗体在材料表中列出。来自不同物种的多种主要抗体可以同时混合。</li>
	<li>将后、前皮肤转移到含150µL 的原发抗体溶液的新井中。在4摄氏度的搅拌机上用温和的混合孵育皮肤。</li>
	<li>第二天, 将后、前皮肤转移到24井板中的新井, 或吸入含有原发抗体的阻塞缓冲液。增加1毫升的 2% HIGS 稀释在 pbs 与 0.2% TX-100 洗涤缓冲器或 2% DS 稀释在 pbs 与 0.2% TX-100 洗涤缓冲器。用3的洗涤缓冲器的变化, 每15分钟清洗皮肤, 在混合器的室温下轻轻搅拌。</li>
	<li>准备二级抗体溶液 (材料表)。在阻塞缓冲器中稀释二次抗体 (10% HIGS 或 10% DS), 并通过0.22 µm 聚偏聚氟 (PVDF) 膜注射器过滤器连接到1毫升注射器, 过滤含有二级抗体的阻塞缓冲器。 注: Alexa 488 或633共轭山羊抗兔 IgG (H + L) 或小鼠 IgG2a Tuj1, alexa 647-共轭山羊抗仓鼠 igg (h + l) 为 PECAM-1, alexa 488 共轭山羊抗兔 igg (h + l) 为 MBP, 和 Alexa 594 共轭大鼠 igg (h + l) 为 CD11b 使用以1:250 稀释在最后的集中8µg/毫升。皮肤用 Cy3-conjugated αSMA 抗体 (1:500 稀释在最后浓度的2-3 µg/毫升) 与这些继发抗体结合而孵化。</li>
	<li>离心溶液在 1.3万 x g 10 分钟内从阻塞缓冲器中去除二次抗体的聚集粒子。 注: 不同种类的荧光共轭二次抗体可同时混合。</li>
	<li>将后、前皮肤转移至含150µL 的二级抗体溶液。在铝箔上包装24井板, 以避免光和孵化的皮肤为 1 h 与温和的混合在一个搅拌机在室温下。</li>
	<li>将后、前皮肤转移到24井板中的新井, 或完全吸管二次抗体溶液。添加1毫升的 2% HIGS 洗涤缓冲器或 2% DS 洗涤缓冲器。</li>
	<li>在铝箔上包裹24井板, 用洗涤缓冲器的3变化每15分钟清洗一次, 在室温下混合搅拌。</li>
</ol>3. 在幻灯片上安装耳皮
<ol><li>将皮肤放在 35 x 10 毫米2培养皿的底部。用低光照显微镜下的细弯钳, 从皮肤内侧小心去除毛发、脂肪组织、结缔组织、灰尘和纤维, 以尽量减少广泛的照片漂白。用 PBS 把皮肤弄湿。</li>
	<li>使用镊子将皮肤转移到粘附显微镜滑动。将后部和前部皮肤与内侧面朝上滑动。用镊子将皮肤平整。</li>
	<li>在液体防褪色安装介质中安装皮肤, 以避免漂白和保存荧光信号。确保没有气泡在皮肤上或周围。</li>
	<li>在皮肤标本上仔细盖上盖玻片, 在室温下将装入的皮样幻灯片放在黑暗中, 让安装介质变得牢固。用指甲油将盖玻片密封到滑轨上, 并将其存放在4摄氏度, 用于长期贮存。</li>
</ol>4. 共焦显微术
<ol><li>为显影设置合适的激光器。本实验采用三激光源 (氩 488 nm、DPSS 561 nm 和 HeNe 633 nm) 共焦显微镜。</li>
	<li>使用顺序扫描工具, 同时激发三重染色样品, 以避免和减少任何重叠。 注意: 图像将使用顺序扫描模式以顺序方式进行。</li>
	<li>10X 目标下的图像。使用平铺扫描工具捕捉整个耳朵皮肤。设置 z 堆栈, 并确保 z 位置覆盖整个耳朵皮肤的厚度。</li>
</ol>

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., James, J., Nam, J., Uchida, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+confocal+microscopy+for+vascular+branching+morphogenesis.">Whole-mount confocal microscopy for vascular branching morphogenesis.</a> Methods Mol Biol. 843, 69-78 (2012).</li><li>Li, W., Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemical+analysis+for+embryonic+limb+skin+vasculature:+a+model+system+to+study+vascular+branching+morphogenesis+in+embryo.">Whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo.</a> J Vis Exp. , (2011).</li><li>Yamazaki, T., 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=Tissue+Myeloid+Progenitors+Differentiate+into+Pericytes+through+TGF-beta+Signaling+in+Developing+Skin+Vasculature.">Tissue Myeloid Progenitors Differentiate into Pericytes through TGF-beta Signaling in Developing Skin Vasculature.</a> Cell Rep. 18, 2991-3004 (2017).</li><li>Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel-dependent+recruitment+of+sympathetic+axons:+looking+for+innervation+in+all+the+right+places.">Vessel-dependent recruitment of sympathetic axons: looking for innervation in all the right places.</a> J Clin Invest. 124, 2855-2857 (2014).</li><li>Li, W., 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=Peripheral+nerve-derived+CXCL12+and+VEGF-A+regulate+the+patterning+of+arterial+vessel+branching+in+developing+limb+skin.">Peripheral nerve-derived CXCL12 and VEGF-A regulate the patterning of arterial vessel branching in developing limb skin.</a> Dev Cell. 24, 359-371 (2013).</li><li>Chang, H., Wang, Y., Wu, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flat+mount+imaging+of+mouse+skin+and+its+application+to+the+analysis+of+hair+follicle+patterning+and+sensory+axon+morphology.">Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology.</a> J Vis Exp. , e51749 (2014).</li><li>Salz, L., Driskell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+Whole+Mount:+A+Novel+Processing+and+Imaging+Protocol+for+Thick,+Three-dimensional+Tissue+Cross-sections+of+Skin.">Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin.</a> J Vis Exp. , (2017).</li><li>Liakath-Ali, 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=Novel+skin+phenotypes+revealed+by+a+genome-wide+mouse+reverse+genetic+screen.">Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen.</a> Nat Commun. 5, 3540 (2014).</li><li>Gunawan, M., 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=The+methyltransferase+Ezh2+controls+cell+adhesion+and+migration+through+direct+methylation+of+the+extranuclear+regulatory+protein+talin.">The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin.</a> Nat Immunol. 16, 505-516 (2015).</li><li>Avci, P., 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=Animal+models+of+skin+disease+for+drug+discovery.">Animal models of skin disease for drug discovery.</a> Expert Opin Drug Dis. 8, 331-355 (2013).</li><li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">Animal models of atopic dermatitis.</a> J Invest Dermatol. 129, 31-40 (2009).</li><li>Wagner, E. F., Schonthaler, H. B., Guinea-Viniegra, J., Tschachler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psoriasis:+what+we+have+learned+from+mouse+models.">Psoriasis: what we have learned from mouse models.</a> Nat Rev Rheumatol. 6, 704-714 (2010).</li><li>Nunan, R., Harding, K. G., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+challenges+of+chronic+wounds:+searching+for+an+optimal+animal+model+to+recapitulate+their+complexity.">Clinical challenges of chronic wounds: searching for an optimal animal model to recapitulate their complexity.</a> Dis Model Mech. 7, 1205-1213 (2014).</li><li>O'Brien, P. D., Sakowski, S. A., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+diabetic+neuropathy.">Mouse models of diabetic neuropathy.</a> ILAR J. 54, 259-272 (2014).</li><li>Yamazaki, T., 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=Whole-Mount+Adult+Ear+Skin+Imaging+Reveals+Defective+Neuro-Vascular++Branching+Morphogenesis+in+Obese+and+Type+2+Diabetic+Mouse+Models.">Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.</a> Sci Rep. 8, (2018).</li></ol>

作者没有什么可透露的。

本协议描述成人耳皮肤的全芒 immunonohistochemical 成像, 用于分析神经血管结构和免疫细胞分布。我们认为, 这种方法有许多实验优势, 研究人员的分支形态发生和模式的周围神经和血管, 以及三维分布的皮肤成分, 包括免疫细胞和头发毛囊.图像的结果可以用成像软件进行量化, 进一步定量分析。
正确地制备耳部皮肤对本议定书的成功至关重要。首先, euthanizing 后应仔细解剖耳朵皮...

皮肤由三层组成: 表皮、真皮和皮下组织。它已被作为一个模型系统来研究干细胞的维持, 分化和形态发生的发展, 以及再生, 肿瘤发生和炎症的成人。皮肤有丰富的血管化和支配, 使周围神经系统和血管系统的发育协调良好。
我们以前已经展示了一个完整的胚胎皮肤成像技术与多重标记研究完整的周围神经和血管, 包括其蜂窝组件1,2,3,4: 血管中的感觉轴突、交感神经轴突、神经的雪旺细胞、内皮细胞、毛细血管和血管平滑肌细胞 (VSMCs)。在血管生成过程中, 一个主要的毛细管网络经过密集的血管重塑, 发展成为一种分层的血管分支网络。在发育的真皮/皮下组织, 动脉分支与周围的感觉神经和静脉形成相邻的动脉。在分层的血管网络被 VSMCs 彻底覆盖后, 交感神经延伸沿和支配大口径血管1,5,6。尽管发展中的神经和血管系统之间的密切联系具有重要意义, 但一个主要问题是解决成人不同病理情况下神经血管网络的发生情况。三维高分辨率成像是必要的, 以了解发病机制, 以及解剖可识别的分支形态发生和模式。
组织切片染色法分析成年小鼠皮肤的神经细胞和血管形态。其他研究还使用了整个影像的皮肤来可视化周围神经和血管, 除了毛囊, 皮脂腺, 和 arrector 毛肌肉7,8,9。然而, 成人皮肤的厚度使得它很难分析整个深度的皮肤。
在本研究中, 我们开发了一种新的高分辨率的成人耳皮肤的全安装成像, 以克服这些挑战。耳朵皮肤是容易接近的解剖和后续整个安装成像的皮肤在其整个深度。因此, 它是一个简单的和高度重复的方法, 可用于比较三维结构的周围神经和血管系统的皮肤, 与综合量化测量。结果表明, 成人皮肤中保留了大口径血管对周围感觉和交感神经的定位。该协议的目标是可视化分支形态发生, 以及周围神经和血管的模式, 以及在各种情况下, 如炎症和成年小鼠模型的细胞水平上的免疫细胞分布。再生.

<table><tbody><tr><td>10 x Phosphate Buffered Saline</td><td>KD Medical</td><td>RGE-3210</td><td>PBS, without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Gibco</td><td>14025-092</td><td>HBSS, with Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA, fixative, diluted in PBS</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td>Detergent</td></tr><tr><td>Normal goat serum</td><td>Gibco</td><td>16210064</td><td>Component of blocking/washing buffer</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immuno research</td><td>017-000-121</td><td>Component of blocking/washing buffer</td></tr><tr><td>Curved fine tweezers</td><td>Dumont</td><td>RS-5047</td><td></td></tr><tr><td>Curved tweezers</td><td>Integra Miltex Vantage</td><td>V918-782, V918-784</td><td></td></tr><tr><td>Filter Unit 0.45 mm</td><td>Thermo Scientific</td><td>157-0045</td><td>For filtration</td></tr><tr><td>1 mL syringe</td><td>Coviden</td><td>8881501400</td><td>For filtration</td></tr><tr><td>Syringe filter Unit 0.22 mm</td><td>Millex-GV</td><td>SLGVR04NL</td><td>For filtration</td></tr><tr><td>ProLong Gold</td><td>Thermo Scientific</td><td>P36934</td><td>Anti-fade mounting medium</td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td>For sealing</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>MZ95</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Photoshop CC 2017</td><td>Adobe</td><td></td><td>Graphics editor software</td></tr><tr><td>Illustrator CC 2017</td><td>Adobe</td><td></td><td>Graphics editor software</td></tr><tr><td>Image J</td><td>NIH</td><td></td><td>Image processing software</td></tr><tr><td>Anti-PECAM-1 (CD31) antibody</td><td>Millipore</td><td>MAB1398Z</td><td>Hamster IgG, vascular endothelial cell marker, 1:300</td></tr><tr><td>Anti-PECAM-1 (CD31) antibody</td><td>BD Pharmingen</td><td>553369</td><td>Rat IgG2a kappa, vascular endothelial cell marker, 1:300</td></tr><tr><td>Anti-&amp;#945;SMA antibody conjugated with cy-3</td><td>Sigma</td><td>C6198</td><td>Mouse IgG2a, vascular smooth muscle cell marker, 1:500</td></tr><tr><td>Anti-EphB1 antibody</td><td>Santa Cruz</td><td>sc-9319</td><td>Goat polyclonal, venous endothelial cell marker, 1:100</td></tr><tr><td>Anti-neuron-specific Class III &amp;#946;-tubulin (Tuj1)</td><td>Abcam</td><td>AB18207</td><td>Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500</td></tr><tr><td>Anti-Tuj1 antibody</td><td>Covance</td><td>MMS-435P</td><td>Mouse IgG2a, pan-axonal marker, 1:500</td></tr><tr><td>Anti-MBP antibody</td><td>Abcam</td><td>AB40390</td><td>Rabbit polyclonal IgG, myelination marker, 1:200</td></tr><tr><td>Anti-Tyrosine Hydroxylase antibody</td><td>Chemicon</td><td>AB152</td><td>Rabbit polyclonal, sympathetic neuron marker, 1:500</td></tr><tr><td>Anti-Peripherin antibody</td><td>Chemicon</td><td>AB1530</td><td>Rabbit polyclonal, peripheral neuron marker, 1:1000</td></tr><tr><td>Anti-CD11b antibody</td><td>Bio-Rad</td><td>MCA74G</td><td>Rat IgG2b, inflammatory cell marker (macrophages), 1:50</td></tr><tr><td>Anti-CD45 antibody</td><td>Thermo Fisher Scientific</td><td>14-0451-85</td><td>Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500</td></tr><tr><td>Anti-CD3 antibody</td><td>Bio-Rad</td><td>MCA1477T</td><td>Rat IgG1, immune cell marker, 1:100</td></tr><tr><td>Anti-CD45R (B220) antibody</td><td>Thermo Fisher Scientific</td><td>14-0452</td><td>Rat IgG2a kappa, inflammatory cell marker, 1:200</td></tr><tr><td>Anti-GFP antibody</td><td>Thermo Fisher Scientific</td><td>A11122</td><td>Rabbit polyclonal, 1:300</td></tr><tr><td>Anti-GFP antibody</td><td>Abcam</td><td>Ab13970</td><td>Chicken polyclonal, 1:500</td></tr><tr><td>Anti-&amp;#946;-gal antibody</td><td>Cappel</td><td>55976</td><td>Rabbit polyclonal, 1:5000</td></tr><tr><td>Anti-RFP antibody</td><td>Abcam</td><td>Ab62341</td><td>Rabbit polyclonal, 1:300</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Alexa 488</td><td>Thermo Fisher Scientific</td><td>A11034</td><td>Rabbit polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-hamster IgG (H+L) Alexa 647</td><td>Jackson Immuno research</td><td>127-605-160</td><td>Hamster polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-rat IgG (H+L) Alexa 594</td><td>Jackson Immuno research</td><td>112-585-167</td><td>Rat polyclonal secondary antibody, 1:250</td></tr><tr><td>Goat anti-mouse IgG2a Alexa 633</td><td>Thermo Fisher Scientific</td><td>A21136</td><td>Mouse IgG2a secondary antibody, 1:250</td></tr></tbody></table>

whole-mount confocal microscopy for adult ear skin: a model system to study neuro-vascular branching morphogenesis and immune cell distribution

在这里, 我们提出了一个完整的成人耳皮肤成像技术的协议, 以研究全面的三维神经血管分支形态发生和模式, 以及免疫细胞分布的细胞水平。对成人组织周围神经和血管解剖结构的分析, 为了解创伤愈合等病理条件下的功能性神经血管接线和神经血管变性提供了一些见解。作为一个高度信息的模型系统, 我们把我们的研究集中在成人耳皮肤, 这是容易被解剖。我们的简单和可重现的协议提供了一个准确的描述整个皮肤的细胞成分, 如周围神经 (感觉轴突, 交感神经轴突, 和雪旺细胞), 血管 (内皮细胞和血管平滑肌细胞) 和炎症细胞。我们相信这项议定书将为调查不同病理条件下成人耳皮肤的形态学异常和周围神经和血管的炎症铺平道路。

成年小鼠后耳皮肤 (图 1A) 和前耳皮肤 (图 1B) 与αSMA (红色)、Tuj1 (绿色) 和 PECAM-1 (蓝色) 的抗体 immunostained。后皮肤 immunostained 研究神经免疫分布, 使用抗体 CD11b (红色) 和 MBP (绿色), 连同 Tuj1 (蓝色) (图 2A)。在单个细胞分辨率 (图 2B) 中检测到 CD11b+炎症细胞 (包括巨噬细胞) 的分?...

Watch this Scientific Journal Video about Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution at JoVE.com

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

在这里, 我们描述了一个高分辨率全安装成像方法在整个成年小鼠耳皮肤, 这使我们能够想象分支形态发生和模式的周围神经和血管, 以及免疫细胞分布。

成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

whole-mount-confocal-microscopy-for-adult-ear-skin-model-system-to

Research

JoVE Journal

Developmental Biology

13.0K 次观看.  National Institutes of Health. 在这里, 我们描述了一个高分辨率全安装成像方法在整个成年小鼠耳皮肤, 这使我们能够想象分支形态发生和模式的周围神经和血管, 以及免疫细胞分布。

视频: Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

科研

生物工程

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 ...

神经科学

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

免疫与感染

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 ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

生物学

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.
Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.
Included in our methods are special considerations made for the cochlea given its 
shape and structure.&#160;Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

小鼠内耳组织的冷冻切片和免疫染色：从胚胎阶段到成人阶段

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is ...

发育生物学

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

医学

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

The ear model of the hairless SKH1-Hrhr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

活体显微镜及血栓诱导在无毛小鼠的耳垂

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

用于单核测序或免疫染色的血管纹显微切割

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统

摘要

探索更多视频

此视频中的章节