Name: generation of 3-d collagen-based hydrogels to analyze axonal growth and behavior during nervous system development
Uploaded: 2019-06-25T13:00:00
Description: Watch this Scientific Journal Video about Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development at JoVE.com

作者感谢汤姆·约汉南的编辑建议和M.塞古拉-费柳的技术援助。这项工作由CERCA方案和加泰罗尼亚大学、大学和企业部大学与研究委员会资助(SGR2017-648)。这项工作由西班牙研究、创新和大学部(MEICO)通过BFU2015-67777-R和RTI2018-099773-B-100、西班牙普里昂网络(西班牙Prionet AGL2017-90665-REDT)和卡洛斯三世研究所资助。PRY-2018-2)。

所有动物实验均根据巴塞罗那大学动物实验伦理委员会(CEEA)的指导方针和规程进行,该研究中的啮齿动物使用协议由巴塞罗那大学欧洲动物实验中心审查和批准。巴塞罗那大学(CEEA批准#276/16和141/15)。
1. 大鼠尾部胶原蛋白的纯化
<ol><li>收集成年斯普拉格-道利大鼠尾巴(8-9周大)后牺牲动物遵循伦理准则,并在95%乙醇冲洗。将 2-4 尾鱼放在冰上 (4 °C),并在过程中将其覆盖在冰上。</li...

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发育的斧子的生长主要是侵入性的,包括ECM降解和重塑。利用这里介绍的程序,研究人员可以获得由自然型I胶原蛋白形成的均质三维基质,其中斧子(或细胞)可以响应转基因细胞在体内分泌的化学梯度。对吸引或抑制性线索(蛋白质、脂质等)梯度的不同斧状反应可以很容易地与特定控制(模拟转染细胞)进行比较。作为优点,我们必须提到,肌腱很容易分离,事实上,它们可能是动物实验的残余物。此外,肌腱?...

神经性斧头,以斧头生长锥结束,通过胚胎的细胞外基质(ECM)在特定的路径上迁移长距离,以达到适当的目标。生长锥体是斧子的远端部分,它专门用于感知细胞1,2的物理和分子环境。从分子的角度来看,生长锥体至少受四种不同的分子机制的引导:接触吸引、化学吸引、接触排斥和由不同斧突引导线索触发的切口3,4,5,6.接触介导过程可在微图案基板上的二维(2D)培养物中进行部分监测(例如,带条纹7、8或点9含有分子)。然而,斧子可以通过从环境中的引导柱细胞中感应到几个有吸引力的和令人厌恶的分子,以非扩散的方式导航到它们的目标。在这里,我们描述了一种简单的三维培养方法,以检查分泌分子是否对开发斧子产生化学吸引力或化学吸引力。
最早的研究旨在确定在三维(3-D)矩阵中使用外植培养物的斧子引导线索的影响,以生成模拟体内条件11、12的梯度。这种方法,连同体内实验,允许识别四个主要的家族的指导线索:内特林,斯利茨,塞马波林,和Ephrins4,5,6。这些分子线索和其他因素13被生长的斧突整合,触发粘附复合物的动态,并通过细胞骨架14、15、16传递机械力。为了在三维培养物中产生分子梯度,用于异形导航,先驱研究人员使用等离子凝固基质17,也用于有机切片制剂18。然而,在1958年,一个新的协议,以产生3D胶原水凝胶报告用于研究与Maximow的设备19,一个培养平台,用于几个研究适合微观观察20。另一项先驱研究报告说,胶原蛋白凝胶作为一种工具,将人类成纤维细胞嵌入到伤口愈合过程中的肌纤维细胞中。同时,Lumsden和Davies应用牛皮毛蛋白的胶原蛋白,分析神经生长因子(NGF)对感觉神经纤维22的引导作用。随着不同公司和实验室开发新的培养平台(例如多孔板),胶原蛋白培养物被适应这些新设备6、23、24、25 ,26.同时,从恩格尔布雷斯-霍尔姆-肖姆-肖姆肿瘤细胞系中提取的ECM物质的提取物也用于商业用途,以扩大这些研究27。
最近,已经开发出几种协议,利用三维水凝胶(例如胶原蛋白、纤维蛋白等),在斧子指导中产生具有假定作用的分子梯度。28.或者,候选分子可以在多孔基质(例如NGF29)中以不同浓度固定,或通过在分泌分子的3-D水凝胶细胞聚集体的小区域内培养产生,从而产生径向梯度4,23,24,25,26 。最后一种可能性将在本协议中解释。
这里介绍的程序是一种简单、快速和高度可重复的方法,基于对胚胎小鼠大脑三维水凝胶培养物的斧头生长的分析。与其他方法相比,该协议非常适合未经培训的研究人员,可在短期培训(1-2 周)后完全开发。在此协议中,我们首先从成年大鼠尾部分离胶原蛋白,以进一步生成三维基质,其中转基因细胞聚集物在胚胎神经元组织前培养。这些细胞聚集体形成候选分子的径向化学梯度,从而引起对生长的斧子的反应。最后,使用相对比显微镜或免疫细胞化学方法,可以轻松评估分子对生长的斧子的影响。

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			<td height="24" style="height:24px;width:455px;">Material</td>
			<td style="width:211px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
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			<td height="22" style="height:22px;">3,3&#8242;-Diaminobenzidine tetrahydrochloride 10 mg tablets (DAB)</td>
			<td>Sigma</td>
			<td>D5905</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="21" style="height:21px;">Adult Sprague-Dawley rats (8 to 9 weeks old)&#160;</td>
			<td>Criffa-Credo, Lyon, France</td>
			<td></td>
			<td style="width:167px;"></td>
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			<td height="22" style="height:22px;">Avidin-biotin-peroxidase complex (ABC)</td>
			<td>Vector Labs</td>
			<td>PK-4000</td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="24" style="height:24px;">B27 serum-free supplement 50x&#160;</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;">Bicinchoninic acid (BCA) protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
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			<td height="21" style="height:21px;">cDNA plasmid vectors</td>
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			<td height="23" style="height:23px;">COS1 cell lines</td>
			<td>ATTC</td>
			<td>CRL-1570</td>
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		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>16325</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">D-(+)-glucose (45% solution in water) for complete Neurobasal medium</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">D-MEM (Dulbecco&#39;s Modified Eagle Medium 1x ) for COS1 culture medium</td>
			<td>Invitrogen</td>
			<td>41966-029</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Dulbecco&#8217;s phosphate buffered saline 10x (without Ca2+ and Mg2+) (D-PBS) for cultures</td>
			<td>Invitrogen</td>
			<td>14200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Ethanol&#160;</td>
			<td>merck</td>
			<td>108543</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ethylenediaminetetraacetic acid dihydrate disodium salt (EDTA)</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fluorescence mounting media (e.g., Fluoromount-G or similar)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>17984-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gelatin powder&#160;</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Glacial acetic acid (Panreac, cat. no. 211008)</td>
			<td>Panreac</td>
			<td>211008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">Hank&#8217;s balanced salt solution&#160;</td>
			<td>Invitrogen</td>
			<td>24020083</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Heat-inactivated foetal bovine serum&#160;</td>
			<td>Invitrogen</td>
			<td>10108-165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Heat-inactivated horse serum&#160;</td>
			<td>Invitrogen</td>
			<td>26050-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hydrogen peroxide (H2O2, 32 to 33% in water)</td>
			<td>Sigma</td>
			<td>316989</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine 200 mM solution (100x) for complete Neurobasal and COS1 medium</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000 Reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Mice pregnant female (embryonic day 12.5 to 16.5; E12.5-16.5)&#160;</td>
			<td>Criffa-Credo, Lyon, France</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modified Minimum Essential Medium Eagle (MEM)</td>
			<td>Invitrogen</td>
			<td>11012-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monoclonal antibody against class III &#946;-tubulin (clone TUJ-1)</td>
			<td>Biolegend</td>
			<td>801201</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-2 supplement 100x&#160;</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal medium&#160;</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Merck</td>
			<td>1,040,051,000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin solution 100x&#160;</td>
			<td>Invitrogen</td>
			<td>15140-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline 10x (PBS) for immunocytochemistry</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibody: biotinylated horse anti-mouse&#160;</td>
			<td>Vector Labs</td>
			<td>BA-2000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-free medium (Opti-MEM)</td>
			<td>Invitrogen</td>
			<td>11058-021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide&#160;</td>
			<td>Panreac</td>
			<td>162712</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate solution 7.5%&#160;</td>
			<td>Invitrogen</td>
			<td>25080-094</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile culture grade H2O&#160;</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TritonTM X-100&#160;</td>
			<td>Sigma</td>
			<td>X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizma base&#160;</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (Trypsin (0.05% (wt/vol) with EDTA (1x)</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:455px;">Equipment</td>
			<td style="width:211px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 large and 1 small curved scissors for dissection&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5-ml conical centrifuge tubes&#160;</td>
			<td>Eppendorf or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-ml conical centrifuge tubes&#160;</td>
			<td>Corning or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 haemostats&#160;&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 small straight dissecting scissors</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200-ml centrifuge tubes for centrifugation</td>
			<td>Nalgene or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200-ml sterile glass conical flasks</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-litre glass beaker</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4- and 6-well culture plates&#160;</td>
			<td>Nunc</td>
			<td>176740 and 140675</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic pipette pumps and disposable 10 ml and 25 ml filter-containing sterile plastic pipettes.</td>
			<td colspan="2">Gilson, Brand, Eppendorf or similar&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automatic pipettes, sterile filter tips and current sterile tips&#160;</td>
			<td>Gilson, Eppendorf or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bench top microcentrifuge with angle fixed rotor&#160;</td>
			<td colspan="2">Eppendorf, Beckman Coulter or similar</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bench top refrigerated centrifuge with swing-bucket rotor (with 1.5, 15 and 50 ml tube adaptors)</td>
			<td colspan="2">Eppendorf, Beckman Coulter or similar</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture incubator at 37 &#186;C, 5% CO2 and 95% air</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis tubing cellulose membrane</td>
			<td>Sigma</td>
			<td>D9402</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis tubing closures&#160;</td>
			<td>Sigma</td>
			<td>Z37101-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable glass pipettes</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope with dark field optics&#160;</td>
			<td>Olympus SZ51 or similar&#160;</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">High-speed refrigerated Beckman Coulter centrifuge or similar with angle fixed rotor</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow hood</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large 100-mm, 60-mm and small 35-mm &#216; cell culture dishes&#160;</td>
			<td>Nunc</td>
			<td>&#160;150679 , 150288 and 150318, respectively</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic stirrer and magnetic spin bars</td>
			<td>IKA or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">McIlwain tissue chopper</td>
			<td>Mickle Laboratory Engineering</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">One pair of fine straight forceps and one pair of curved forceps&#160;</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades for the tissue chopper</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpels (number 15 and 11)</td>
			<td></td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two pairs of fine spatulas for transferring collagen and tissue pieces</td>
			<td>Fine tools Instruments or similar</td>
			<td></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

generation of 3-d collagen-based hydrogels to analyze axonal growth and behavior during nervous system development

该协议使用天然I型胶原蛋白生成三维(3-D)水凝胶,用于监测和分析斧状生长。该方案的核心是在大鼠尾巴肌腱衍生的I型胶原蛋白中形成的3D水凝胶内培养小块胚胎或产后啮齿动物大脑,具有特定的孔隙度。组织片在水凝胶内培养,并面对特定的脑碎片或转基因细胞聚集物,以产生和分泌适合在多孔基质内形成梯度的分子。该协议的步骤简单且可重现,但包含开发过程中要仔细考虑的关键步骤。此外,在免疫细胞化学方法固定后,可直接使用相对比显微镜或单光/多光子荧光显微镜监测和分析生长的斧子的行为。

在这里,我们提出了一个广泛可访问的方法,研究胚胎小鼠神经系统的3D水凝胶胶原蛋白培养物的斧头生长。为此,我们从成年大鼠尾部分离出胶原蛋白,以生成三维基质,其中我们培养的转基因细胞聚集体,表达Netrin-1或Sema3E,与胚胎神经元组织(例如,海马的CA区域)对抗。这些细胞聚集体在胶原蛋白基质内形成候选分子的径向分布梯度。最后,为了评估神经元对不同分子的反应,我们使用免疫细胞化学方法(例...

Watch this Scientific Journal Video about Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development at JoVE.com

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

在这里,我们提供了一个方法,用于分析在3D矩阵中生长斧子的行为,模仿它们的自然发育。

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

该方法有助于分析某些分子在神经系统发育过程中在弓形指导和神经元迁移中的作用。该技术的主要优点是，结果分析是快速和简单的，不需要复杂的软件，这将意味着大量的培训研究人员。演示该程序将是米里安塞古拉-费利乌，一个技术人员从我们的实验室和我。将20万个COS-1细胞开始放入35毫米培养皿中，并在细胞培养培养箱中用完整的培养，在一夜之间读取70至80%的汇合。第二天，用DNA对候选分子进行转染，使用基于脂体体的转染方法对候选分子进行编码。首先将250微升的血清自由介质和1至2微克DNA混合在1.5毫升离心管中，在室温下孵育5分钟。要准备脂质体管，加入240微升的血清自由培养剂和10微升脂体转染试剂，并在室温下孵育5分钟。孵育后，将DNA管的含量加入唇管。在室温下轻轻混合和孵育15分钟。用1.5毫升的血清自由培养剂取代培养细胞上的培养。慢慢加入DNA脂质体混合物，并明智地滴入细胞。在二氧化碳细胞培养孵化器中孵育三小时。完成孵育后，用完整的培养培养本取代培养本，在孵化器中孵育。第二天，用0.1摩尔杜尔贝科的PBS冲洗细胞。每道菜加入800微升尝试素EDTA，在二氧化碳培养箱中孵育5至15分钟。将两毫升完整培养培养的分离细胞收集到15毫升的离心管中，并加入10毫升的同一培养。在4摄氏度下将细胞在130倍g下离心5分钟。取出上一液，将含有COS-1细胞的颗粒保留在冰上。制备胶原蛋白工作混合物后，将100至150微升的胶原蛋白混合物加入转染细胞的颗粒中，然后上下移液轻轻混合。将 45 至 50 微升胶原蛋白细胞颗粒混合物扩散到 35 毫米培养皿上，形成一个均匀的胶原蛋白细胞带，长约 1 至 1.5 厘米。将培养皿在37摄氏度和5%的二氧化碳下放在培养箱中，直到观察到凝胶。在第二个培养盘中准备第二个包含控制细胞的条带，并放在培养箱中。凝胶完成后，将三到四毫升37摄氏度的加热COS-1完整的培养培养剂添加到每盘含有凝胶胶原蛋白细胞条的培养皿中，并将其放在培养箱中。使用细手术刀或组织斩波器切割胶原蛋白细胞条，生成长度为 400 到 500 微米的小块方形片段。将所有部分从相同的转染条件转移到含有三到三点五毫升神经元培养素的培养皿。在解剖显微镜下检查碎片的质量，将盘子放在培养箱中。使用剪刀在胚胎第16.5天从牺牲怀孕的雌性大鼠的腹腔切割胚胎角，并把它们放入含有冷HSSG缓冲液的大培养皿中。将盘子放在层流罩中，用直钳提取胚胎，并放入含有冷 HBBSg 的新盘中。用小钳子，去除皮肤，然后用弯曲和直钳，仔细解剖大脑，放入含有冷HPSSg的盘子。在解剖显微镜下，用手术刀或细剪刀将大脑沿着中线切成两半，以分离两个半球。然后，取出死头和细钳，从脑片中去除脑和血管。使用 100% 乙醇清洁组织切碎器的所有部分，特别是 PTFE 切割板和剃须刀刀片，并在紫外线照射下将组织切碎器放在层流罩中 15 分钟。将每个组织片转移到组织切碎器的切割板。组织被切碎后，从35毫米培养皿中转移，里面装满了三到四毫升完整的NCM。使用细钨针，完成组织解剖在完整的NCM。在解剖显微镜下检查获得的切片的质量，确保在暗场光学元件中可以清楚地识别这些层，并用钨针解剖感兴趣的区域。将优质片件在二氧化碳培养箱中保持完整的 NCM 介质。视觉演示对于与共同文化准备相关的步骤至关重要，这些步骤有助于单词理解过程。将几个无菌的四孔培养板放在层流罩中，并像以前一样准备胶原蛋白工作混合物。将15至20微升水凝胶混合物加入到每孔底部，形成圆形胶原蛋白基。将盘子放在培养箱中，直到观察到完全的凝胶。并检查凝胶胶原蛋白的质量。使用移液器，将一小块 COS-1 细胞聚合转移到水凝胶基座上，然后使用移液器将组织块放在同一基座上，靠近一块细胞聚合体，距离一个外植大小。在冰上制备新的工作胶原蛋白混合物后，轻轻移液15至20微升这种新的混合物，以覆盖外植和细胞聚集体，使三明治像水凝胶培养。使用细钨针重新定向外植，使其面对500至600微米的细胞聚集。将板返回培养箱，直到观察到凝胶，然后加入0.5毫升的完整NCM，辅以2%B27补充剂，并在培养箱中保持培养物36至48小时。当海马轴与Netrin-1对抗时，它们优先地向Netrin-1的来源发展，表明Netrin-1对于这些轴子起到化学吸引力的分子的作用。在控制条件或模拟转染中，所有轴子均径向增长，没有任何方向偏好。当海马轴与Sema3E分泌细胞对峙时，它们中的大多数生长在细胞聚集物的对面，表明Sema3E充当这些轴子的化疗排斥分子。轴向和定量方法的原理图表示表明，在控制条件下，轴子在近位象限和近面象限中均等分布，以径向增长显示。这表示近近近近地或 PD 比率为 1。当外植显示近位象限中轴子数量与近方位相比增加时，表示化疗具有吸引力，PD 比大于 1。当轴子的数量在近象限中高于近位象限时，表示化疗排斥，PD比率小于 1。按照这种方法，研究人员可以获取有关某些分子在神经元发育过程中作用的基本信息，从而使我们能够建立一个起点，进一步探索这些功能。虽然这项技术在神经元发育中很有用，但它也可以作为策略应用于药理筛选、血管生成和组织工程。

Research

JoVE Journal

Neuroscience

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Mechanical Manipulation of Neurons to Control Axonal Development

机械操纵控制神经元轴突的发展

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Designing and Implementing Nervous System Simulations on LEGO Robots

LEGO机器人神经系统仿真设计和实现

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...