Name: toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology
Uploaded: 2018-07-03T15:00:00
Description: Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

作者希望 thankthe 詹金斯显微镜设施在怀俄明大学为他们的帮助, 并且布什曼实验室的成员, 凯利 Roballo, 海登真实, Wupu Osimanjiang 并且 Subash Dhunghana, 为动物关心的协助。这份出版物是由国立卫生研究院国立医学研究院的一个机构发展奖 (2P20GM103432) 授予的。

本项目使用成人大大鼠, 所有程序均经怀俄明州大学动物保育和使用委员会批准。
1. 手术和体内神经固定
注: 本协议中使用的所有材料和设备的供应商信息列在材料表中。
注:体内神经固定用于保存组织并减少在死亡时间和神经收集之间可能发生的结构退化。在体内组织固定是一种标准的做法, 以制备神...

<ol>
	<li>Zacchigna, S., Ruiz de Almodovar, C., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+between+angiogenesis+and+neural+development:+What+small+animal+models+can+tell+us.">Similarities between angiogenesis and neural development: What small animal models can tell us.</a> Current Topics in Developmental Biology. 80, 1-55 (2008).</li><li>Grinsell, D., Keating, C. P. <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+reconstruction+after+injury:+A+review+of+clinical+and+experimental+therapies.">Peripheral nerve reconstruction after injury: A review of clinical and experimental therapies.</a> BioMed Research International. 2014, 1-13 (2014).</li><li>Chen, M. B., Zhang, F., Lineaweaver, 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=Luminal+fillers+in+nerve+conduits+for+peripheral+nerve+repair.">Luminal fillers in nerve conduits for peripheral nerve repair.</a> Annals of Plastic Surgery. 57 (4), 462-471 (2006).</li><li>Scipio, F., Raimondo, S., Tos, P., Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+paraffin-embedded+myelin+sheath+staining+with+osmium+tetroxide+for+light+microscope+observation.">A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observation.</a> Microscopy Research and Technique. 71, 497-502 (2008).</li><li>Raimondo, S., Fornaro, M., Di Scipio, F., Ronchi, G., Giacobini-Robecchi, M. G., Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5:+Methods+and+protocols+in+peripheral+nerve+regeneration+experimental+research:+part+II-morphological+techniques.">Chapter 5: Methods and protocols in peripheral nerve regeneration experimental research: part II-morphological techniques.</a> International Review of Neurobioly. 87, 81-103 (2009).</li><li>Carriel, V., Garzon, I., Alaminos, M., Campos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+myelin+sheath+and+collagen+reorganization+pattern+in+a+model+of+peripheral+nerve+regeneration+using+an+integrated+histochemical+approach.">Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach.</a> Histochemistry and Cell Biology. , 709-717 (2011).</li><li>Sridharan, G., Shankar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toluidine+blue:+A+review+of+its+chemistry+and+clinical+utility.">Toluidine blue: A review of its chemistry and clinical utility.</a> Journal of Oral and Maxillofacial Pathology. 16, 251-255 (2012).</li><li>Epstein, J. B., Scully, C., Spinelli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toluidine+blue+and+Lugol's+iodine+application+in+the+assessment+of+oral+malignant+disease+and+lesions+at+risk+of+malignancy.">Toluidine blue and Lugol's iodine application in the assessment of oral malignant disease and lesions at risk of malignancy.</a> Journal of Oral Pathology &amp; Medicine. 21, 160-163 (1992).</li><li>Carriel, V., Garzon, I., Alaminos, M., Cornelissen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+assessment+in+peripheral+nerve+tissue+engineering.">Histological assessment in peripheral nerve tissue engineering.</a> Neural Regeneration Research. 9, 1657-1660 (2014).</li><li>Dykstra, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual of applied techniques for biological electron microscopy. , 257 (1993).</li><li>Vleggeert-Lankamp, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+evaluation+methods+in+the+assessment+of+peripheral+nerve+regeneration+through+synthetic+conduits:+A+systematic+review.">The role of evaluation methods in the assessment of peripheral nerve regeneration through synthetic conduits: A systematic review.</a> Journal of Neurosurgery. 107 (6), 1168-1189 (2007).</li><li>Williams, P., Wendell-Smith, C. P., Finch, A., Stevens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+uses+and+methods+of+processing+of+fresh+frozen+sections+of+peripheral+nerve.">Further uses and methods of processing of fresh frozen sections of peripheral nerve.</a> Journal of Cell Science. 3 (69), 99-105 (1964).</li><li>Castro, J., Negredo, P., Avenda&#241;o, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+composition+of+the+rat+sciatic+nerve+and+its+modification+during+regeneration+through+a+sieve+electrode.">Fiber composition of the rat sciatic nerve and its modification during regeneration through a sieve electrode.</a> Brain research. , 65-77 (2008).</li><li>Raimondo, S., Fornaro, M., Di Scipio, F., Ronchi, G., Giacobini-Robecchi, M. G., Geuna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+and+protocols+in+peripheral+nerve+regeneration+experimental+research:+Part+II-morphological+techniques.">Methods and protocols in peripheral nerve regeneration experimental research: Part II-morphological techniques.</a> International review of neurobiology. 87, 81-103 (2009).</li><li>Bozkurt, A., Lassner, F., O'Dey, D., Deumens, R., B&#246;cker, A., Schwendt, T., Janzen, C., Suschek, C. V., Tolba, R., Kobayashi, E., Sellhaus, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microstructured+and+interconnected+pore+channels+in+a+collagen-based+nerve+guide+on+axonal+regeneration+in+peripheral+nerves.">The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves.</a> Biomaterials. 33 (5), 1363-1375 (2012).</li><li>Weis, J., Brandner, S., Lammens, M., Sommer, C., Vallat, 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=Processing+of+nerve+biopsies:+A+practical+guide+for+neuropathologists.">Processing of nerve biopsies: A practical guide for neuropathologists.</a> Clinical Neuropathology. 31 (1), 7-23 (2012).</li><li>Battiston, B., Tos, P., Geuna, S., Giacobini-Robecchi, M. G., Guglielmone, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+repair+by+means+of+vein+filled+with+muscle+grafts.+II.+Morphological+analysis+of+regeneration.">Nerve repair by means of vein filled with muscle grafts. II. Morphological analysis of regeneration.</a> Microsurgery. 20 (1), 37-41 (2000).</li><li>Bhattacharyya, T. K., Thomas, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+staining+methods+for+resin-embedded+peripheral+nerve.">Comparison of staining methods for resin-embedded peripheral nerve.</a> Journal of Histotechnology. 27, 161-164 (2004).</li><li>Zbaeren, J., Zbaeren-Colbourn, D., Haeberli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+immunohistochemistry+on+improved+glycol+methacrylate-resin+sections.">High-resolution immunohistochemistry on improved glycol methacrylate-resin sections.</a> Journal of Histotechnology. 30 (1), 27-33 (2007).</li><li>Alturkistani, H. A., Tashkandi, F. M., Mohammedsaleh, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+stains:+A+literature+review+and+case+study.">Histological stains: A literature review and case study.</a> Global Journal of Health Science. 8 (3), 72-79 (2016).</li><li>Ezra, M., Bushman, J., Shreiber, D., Schachner, M., Kohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+and+nonporous+nerve+conduits:+the+effects+of+a+hydrogel+luminal+filler+with+and+without+a+neurite-promoting+moiety.">Porous and nonporous nerve conduits: the effects of a hydrogel luminal filler with and without a neurite-promoting moiety.</a> Tissue Engineering Part A. (9-10), 818-826 (2016).</li><li>Bhatnagar, D., Bushman, J. S., Murthy, N. S., Merolli, A., Kaplan, H. M., Kohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+as+a+stabilization+strategy+in+peripheral+nerve+repair+when+using+porous+nerve+guidance+conduits.">Fibrin glue as a stabilization strategy in peripheral nerve repair when using porous nerve guidance conduits.</a> Journal of Materials Science: Materials in Medicine. 28 (5), 79 (2017).</li></ol>

对周围神经损伤和再生的形态学结构的检查是研究的13个经常科目。在本协议中, 我们描述了获取高品质图像的步骤, quantifications 利用大鼠坐骨神经组织嵌入树脂块和染色甲苯胺蓝的组织学数据。该技术提供了一种神经形态学的图像, 可以通过测量轴突数、髓鞘形成程度、浸润性纤维化组织的存在和健康神经来量化神经再生。虽然图像显示切片和处理未受伤的神经作为样本, 同样?...

周围神经延伸整个身体, 支配靶组织与马达或感觉轴突1。由医疗失调和外伤引起的周围神经缺损是一个重大的公共卫生问题, 对2、3的经济影响很大。尽管评估周围神经损伤的结果和了解神经再生的进展, 传统的方法, 如神经组织学和染色技术是定性和定量评估神经健康的基本工具作为最终的结果测量动物模型或切除人体组织。这通常与周围神经功能的电生理测量成配对, 在那里, 形态计量学可以揭示功能性神经再生的原因或没有发生。
甲苯胺蓝染色的树脂嵌入半薄外围神经切片是一种专门的方法, 以成像髓神经纤维, 提供高质量和清晰的详细图像的神经结构4,5,6.甲苯胺蓝是一个耐热 metachromatic 染色, 发现的威廉亨利珀在 1856年7, 并已用于几个医疗应用8。甲苯胺蓝染色的周围神经部分获得的树脂嵌入神经段允许清晰的可视化神经结构。使用锇毒气后固定4,9可增强髓鞘结构的可视化。锇毒气是一种有毒的氧化剂和脂质固定剂, 与脂质中的双键相互作用, 导致了明显的富含脂质的髓鞘10。然而, 锇毒气是有毒的, 昂贵的, 需要更长的神经段的孵化, 并不总是使用。
对周围神经形态学的可视化处理和染色方法进行了研究;石蜡, 低温切片, 环氧树脂嵌入神经切片后, 以甲苯胺蓝或苯二胺溶液染色, 用于定量测定周围神经再生11,12 的形态学变化..这些方法各自有各自的优势和产生的关键数据的轴突, 髓鞘厚度, 轴突直径, 轴突直径髓纤维直径 (g 比) 11,13,14,15.
该协议中的树脂嵌入的主要区别是它促进获得1-2 微米厚度剖面由于树脂的硬度, 同时保持神经的组织学质量。这些稀薄的部分, 相对于从石蜡嵌入获得的4-5 微米厚度部分, 提供了更高的分辨率的外围神经部分, 允许更准确的量化轴突髓鞘形成, 如 g 比, 这不能从较厚的部分16获得。虽然低温切片可用于获得1-2 µm 剖面, 但我们的经验是, 很难获得部分没有大量的大裂缝。这些破裂的部分可能导致不准确计数的轴突的数量和方面的髓鞘形成。
除了甲苯胺蓝染色17, 银染色法18和马尾松的三色染色4也可以用来显示神经轴突。然而, 使用树脂嵌入的大鼠中位神经切片染色的苏木精和三色或马尾的, 显示微弱的髓鞘和无法识别的结构, 而甲苯胺蓝染色显示明确的髓鞘图像, 容易能够量化4。尽管有一定的局限性, 甲苯胺蓝染色的树脂嵌入周围神经是一个宝贵的技术, 可用于当高分辨率图像的神经形态学需要。
树脂嵌入的主要缺点是它是费时的, 并且不允许同一个组织的染色由于抗原检索的困难与石蜡和被冻结的嵌入的部分技术相比。因此, 一般不可能利用相同的组织为染色, 通过树脂嵌入处理甲苯胺蓝染色。虽然在这里不使用, 如果在树脂嵌入部分需要免疫组化, 使用乙二醇甲基丙烯酸酯嵌入树脂允许免疫组化在组织切片, 但它是相对昂贵的19。这可以通过将周围神经分割成分开的部分来减轻, 有些用于植入树脂, 或者在固定后直接染色。
甲苯胺蓝染色的树脂嵌入周围神经的过程, 如大多数病理学分析, 可分为五个阶段, 包括固定, 脱水, 植入, 切片, 染色20。本实验旨在为应用树脂嵌入甲苯胺蓝染色的大鼠坐骨神经切片提供一种实用的方案和实践指导, 以获得高质量的图像。

<table border="0" cellpadding="0" cellspacing="0" width="1092">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dulbecco&#39;s Phosphate Buffer Saline</td>
			<td style="width:216px;">Gibco</td>
			<td style="width:137px;">14200-075</td>
			<td style="width:384px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Glutaraldehyde Solution</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">G6257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Paraformaldehyde</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Phosphate Monobasic Monohydrate</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Toluidine Blue O</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">T3260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Tetraborate Decahydrate</td>
			<td style="width:216px;">Acros Organics</td>
			<td style="width:137px;">205950010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Isoflurane</td>
			<td style="width:216px;">Piramal</td>
			<td style="width:137px;">NDC 66794-013-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Epoxy Embedding Medium Kit</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">45359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Hydroxide Solution</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">72068</td>
			<td>To adjust Trump&#39;s fixative pH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Acetone</td>
			<td style="width:216px;">Fisher Chemical</td>
			<td style="width:137px;">170942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Osmium Tetroxide Solution</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">75632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">VWR Micro Slides, Superfrost Plus</td>
			<td style="width:216px;">VWR</td>
			<td style="width:137px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microscope Cover Glass</td>
			<td style="width:216px;">Fisher Scientific</td>
			<td>12545102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Pelco Embedding Cast</td>
			<td style="width:216px;">Fisher Scientific</td>
			<td>NC9671811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Glass Knife Maker</td>
			<td style="width:216px;">RMC Products</td>
			<td style="width:137px;">GKM-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ultramicrotome</td>
			<td style="width:216px;">RMC Products</td>
			<td style="width:137px;">MT-XL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">15 mL Conical Tube</td>
			<td style="width:216px;">Falcon</td>
			<td>ISO 9001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Eppendorf 1.5 mL microcentrifuge tubes</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td>T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">4 mL Glass Vial</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td>854190</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">Razor Blades</td>
			<td style="width:216px;">VWR</td>
			<td style="width:137px;">55411-050</td>
			<td>For trimming resin block</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">Perfect Loop</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
			<td style="width:137px;">70944</td>
			<td>For picking up thin resin sections</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Ultra Glass Knife Strips 6.4 mm x 25 mm x 400 mm</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
			<td style="width:137px;">71012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">100 Watt Oven</td>
			<td style="width:216px;">Millipore&#160;</td>
			<td style="width:137px;">6350115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Whatman Filter Paper</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td>WHA10010155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">3 mL plastic pipette</td>
			<td style="width:216px;">Sigma-Aldrich</td>
			<td style="width:137px;">Z331740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Micro-surgical Kit</td>
			<td style="width:216px;">World precision instruments</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Olympus fluorescence microscope</td>
			<td style="width:216px;"></td>
			<td style="width:137px;"></td>
			<td>Dual CCD Color and Monochrome Camera, DP80</td>
		</tr>
	</tbody>
</table>

toluidine blue staining of resin-embedded sections for evaluation of peripheral nerve morphology

周围神经延伸整个身体, 支配靶组织与马达或感觉轴突。由于广泛的分布, 周围神经经常因外伤或疾病而受损。由于对周围神经损伤的动物模型、功能和再生方法和策略进行了评估, 分析周围神经的形态计量已成为一个重要的终端结果测量。甲苯胺蓝染色神经横断面从树脂嵌入神经切片是一种重现性的方法, 定性和定量评估周围神经, 使可视化的形态学数字的轴突和程度髓鞘形成.这项技术, 和其他许多组织学方法一样, 很难用标准的书面协议来学习和掌握。因此, 本出版物的目的是强调书面协议, 以甲苯胺蓝染色的周围神经与录像的方法, 利用从大鼠的坐骨神经。在本协议中, 我们描述了在体外周神经固定和组织收集, 并后固定与2% 锇毒气, 嵌入神经在环氧树脂, ultramicrotome 切片的神经1-2μm 厚度。神经部分然后转移到玻璃滑动和染甲苯胺蓝色, 在之后他们定量地和定性地被评估。显示了最常见问题的例子, 以及缓解这些问题的步骤。

树脂嵌入周围神经部分染色甲苯胺蓝允许准确的组织学数据 quantifications。过程概述 (图 2)。在树脂培养基中嵌入的坐骨神经部分和甲苯胺蓝染色显示清晰的图像, 最佳分辨率 (图 3)。神经损伤可引起神经形态学结构的许多变化, 如神经纤维、轴突直径和髓鞘厚度的变化。该方法能保持神经结构的自然形态, 有利于测量轴突直径与总纤维直径...

Watch this Scientific Journal Video about Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology at JoVE.com

Toluidine Blue Staining of Resin-Embedded Sections for Evaluation of Peripheral Nerve Morphology

在这里, 我们提出了一个通过获取和染色1-2 µm 段与甲苯胺蓝可视化周围神经精细结构的协议

甲苯胺蓝染色树脂嵌段评价周围神经形态学

Peripheral nerves extend throughout the body, innervating target tissues with motor or sensory axons. Due to widespread distribution, peripheral nerves ...

This method could help answer key questions in the fields of peripheral nerve injury and regeneration such as what is the morphometry of a peripheral nerve, how many axons are present, what is the state of myelination, and is there fibrotic tissue present? The main advantage of this technique is that it allows for high-resolution visualization of nerve features. Begin by placing an anesthetized rat in the prone position on the top of a dissection mat.Place a nose cone to maintain anesthesia with 1-2%isoflurane. To ensure adequate depth of anesthesia test the animals for pedal withdrawal and palpebral reflexes of the hind feet. Once an appropriate depth of anesthesia is achieved, shave the hair of the hind limbs and sterilize the shaved areas with 70%ethanol.Palpate the femur to identify the proper location for the incision. The femur is located just proximal to the most accessible segment of the sciatic nerve. After making a two to three centimeter incision in the skin along the hind limb from the knee up to the greater trochanter locate the plane between the biceps femoris muscle and the gluteus maximus muscle.Then use microdissection scissors to separate the underlying fascia and expose two to three centimeters of the underlying sciatic nerve. Use a retractor to widen the gap between the two muscles. With fine forceps and iris scissors carefully separate the nerve from the surrounding connective tissue, taking great care not to compress or cut the nerve.Next, cover the exposed nerve with Trump's Fixative and let sit for 10 minutes. If necessary, place gauze under the hind leg to improve the angle so that more fixative can be added into the cavity. Remove the fixative after 10 minutes and repeat this step two more times.Under the dissecting microscope cut the sciatic nerve from both sides using fine dissection scissors, making sure not to stretch or pinch the nerve. Immediately put the nerve sections in 15 mL tubes containing Trump's Fixative and fix at four degrees Celsius for one week, changing the fixative every 48 hours. Post-fixation, carefully remove any remaining fat and connective tissues from the nerve, and use a sharp scalpel to cut the nerve into segments approximately five millimeters in length.Nerve segments can be separated into different labeled tubes. Immerse nerve segments in freshly prepared 2%osmium tetroxide for two hours. Using an epoxy embedding kit, prepare the final embedding mixture.Mix the epoxy embedding medium with DDSA solution and mix the epoxy embedding medium with NMA solution. Mix both solutions for at least 20 minutes with a magnetic stirrer. Immediately before use, combine the accelerator DPM to solution B and then combine this with solution A such that the accelerator's final proportion is between one point five and 2%of the total volume.Transfer the nerve segments to one point five milliliter tubes and after washing with PBS, start the dehydration process by the replacing the PBS with increasing concentrations of acetone and distilled water for 10 minutes each then in 100%acetone three times for 10 minutes each. One critical step is the tissue dehydration. Water and resin will not interact, so any water remaining in the tissue will not be infiltrated by the resin leaving holes in the section devoid of histological qualities.For resin infiltration, first place the nerve segments in a one-to-one mix of embedding mixture and 100%acetone for 30 minutes. After 30 minutes, transfer the segments to a two-to-one mix of embedding mixture and 100%acetone for 30 minutes. Place the nerve segements in silicone rubber embedding molds and gently add the resin on top of nerves, making sure to cover the whole nerve segment while avoiding air bubbles.Leave the resin to polymerize at 60 degrees Celsius overnight. Place a resin block with embedded nerve into the ultramicrotome holder with the trapezoidal side facing up. While viewing through the ultramicrotome scope, use a single-edged blade to trim excess resin surrounding the nerve tissue.Do not penetrate the longitudinal surface of the nerve segment which is recognizable by its dark staining by osmium tetroxide. With a plain glass knife on the ultramicrotome make multiple cross sections to expose a uniform cross-section surface of the nerve. Once this is done, switch to a glass knife with the boat filled with distilled water at room temperature and adjust the ultramicrotome to one to two microns to cut thin sections.Use a metal loop to transfer floating thin sections from the glass knife boat to a drop of deionized water on a glass slide. Another critical step is during the transfer of thin section from the glass knife to slide. These thin sections are very fragile and can break or fold.After sectioning, dry the sections on the slides by passing a slide over a flame several times, making sure not to overheat the sections. Slides can be stored at room temperature for several days if not immediately staining with toluidine blue. To stain the nerve sections, use a plastic pipette or micropipetter to add a drop of toluidine blue solution on the top of the nerve sections.After 20 to 30 seconds, rinse off all toluidine blue solution excess by gently dipping the slides into a jar of deionized water, repeating three to four times until sections are clear. Dry the slides for at least 15 minutes at 60 degrees Celsius or overnight at room temperature. After drying, add regular mounting medium and cover the sections with a coverslip.Finally, examine the mounted sections under a light microscope. A 100X oil immersion lens is recommended for detailed images to calculate g-ratios. Sciatic nerve sections embedded in resin medium and stained with toluidine blue showed clear images with optimal resolution at increasing magnification from 10X to 20X, 40X, and 60X.This method preserves nerve structure and allows for high-resolution, high-magnification imaging which facilitates the measurement of several parameters such as the g-ratio, which is the ratio of axon diameter, indicated by the arrow labeled B, to the total fiber diameter, labeled A.A variety of factors can lead to less than optimal peripheral nerve sections, including the presence of cracks in the section due to improper handling of the nerve. Holes can also occur in the section due to insufficient dehydration. Another possible error in the procedure is folding of the sections, which can be remedied by using inoculating loops for section transfer onto the glass slide.Following this procedure other methods like axon quantification can be performed to answer additional questions like what's the degree of regeneration in a regenerative nerve? After watching this video you should have a good understanding of how to obtain one to two micron peripheral nerve sections to assess nerve morphometry.

Research

JoVE Journal

Neuroscience

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

科研

神经科学

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Optogenetic Stimulation of the Auditory Nerve

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

间接神经元的星形胶质细胞共培养试验：一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Targeting Alpha Synuclein Aggregates in Cutaneous Peripheral Nerve Fibers by Free-floating Immunofluorescence Assay

通过自由浮动免疫荧光测定,在皮外神经纤维中定位阿尔法核糖核酸聚合体

To date, for most neurodegenerative diseases only a post-mortem histopathological definitive diagnosis is available. For Parkinson&#39;s disease (PD), the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

大鼠慢性植入的外周神经刺激电极的制备

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...