Name: high-resolution 3d imaging of rabies virus infection in solvent-cleared brain tissue
Uploaded: 2019-04-30T13:00:00
Description: Watch this Scientific Journal Video about High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue at JoVE.com

作者感谢托马斯·梅滕莱特和维雷纳·特·坎普批判性地阅读了手稿。这项工作得到了梅克伦堡西波美拉尼亚联邦卓越倡议和欧洲社会基金(ESF)格兰特·科因费克特(ESF/14-BM-A55-0002/16)的支持,以及弗里德里希-勒弗勒研究所(Ri-0372)。

使用RABV感染,PFA固定存档脑材料。各自的动物实验研究由国家农业、食品安全和渔业办公室负责的动物护理、使用和道德委员会(LALFF M-V)进行评估,并获得许可。7221.3-2.1-002/11(小鼠)和7221.3-1-068/16(雪铁龙)。根据批准的指南进行动物实验中使用的一般护理和方法。
注意:本议定书使用各种有毒和/或有害物质,包括PFA、甲醇(MeOH)、过氧化氢(H2O2)、阿齐德钠(NaN3)...

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近年来组织清除技术的复苏和进一步发展2、3、4、5、6、7、89,10,11,12,13,<sup class="x...

传统的组织学技术主要依靠器官组织的薄部分,这本质上只能提供对复杂 3D 环境的 2D 见解。虽然原则上是可行的,但串行薄截面的 3D 重建需要苛刻的技术管道,以便对采集的图像1进行切片和后续的硅对齐。此外,在微缩切片后无缝重建 z 体积至关重要,因为机械和计算伪影可能由于非重叠图像平面、染色变化和物理物理造成的图像配准不理想而保留破坏组织,例如,微托姆刀片。相比之下,对完整厚组织样本进行纯光学切片允许获取重叠的图像平面(过采样),从而有助于 3D 重建。这反过来又对分析复杂细胞群(例如,周围胶质和免疫细胞的神经元网络)中的感染过程非常有益。然而,厚组织部分的固有障碍包括光散射和有限的抗体渗透到组织。近年来,开发和优化了各种技术,以克服这些问题2,3,4,5,6,7,8 ,9,10,11,12,13.基本上,目标组织通过处理水性2、3、4、5、6、7进行光学透明化 ,8,9或有机溶剂基10,11,12,13溶液。引入 3DISCO (溶剂清除器官的 3D 成像)11、12及其后续 uDISCO(溶剂清除器官的最终 3D 成像)13提供了一个相对快速、简单且廉价的工具,出色的清算能力。清除协议的主要成分是有机溶剂特-丁醇(TBA)、苯基醇(BA)、苯甲酸酯(BB)和二苯醚(DPE)。iDISCO(溶剂清除器官的免疫标记支持3D成像)14的开发和添加,这是一种兼容的免疫染色方案,与现有方法形成另一个优势,使抗原的深层组织标记得以实现以及免疫染色样本的长期储存。因此,iDISCO14和 uDISCO13的组合允许使用传统的 CLSM 在大组织部分(高达 1 mm)对抗体标记蛋白进行高分辨率成像。
在所有三个维度中保存器官的复杂结构对脑组织尤其重要。神经元构成一个非常异质的细胞亚群,其基于其神经质投影的3D形态高度多样化(由马斯兰15审查)。此外,大脑由多个隔间和子隔间组成,每个隔间和子隔间由不同的细胞亚群及其比例组成,包括胶质细胞和神经元(由冯·巴托尔德等人审查)。作为一种神经性病毒,狂犬病病毒(RABV,由Fooks等人17审查)主要感染神经元,利用他们的运输机械沿着轴向方向从感染的主要部位到达中枢神经系统(CNS)。此处描述的协议(图1A)允许在从受感染的脑组织获得的大型、连贯的图像堆栈中,对RABV和RABV感染的细胞进行免疫染色辅助检测和可视化。这样就可以对感染环境进行无偏见的 3D 高分辨率评估。它适用于各种物种的脑组织,可在固定后或长期储存在甲醛(PFA)中的样品后立即进行,并允许对染色和清除的样品进行数月的储存和再成像。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Reagents</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Benzyl alcohol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">41218</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Benzyl benzoate</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:160px;">BB6630-500ML</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dimethyl sulfoxide</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">4720.2</td>
			<td style="width:297px;">Various buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Diphenyl ether</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:160px;">240834-100G</td>
			<td style="width:297px;">Clearing reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">DL-&#945;-Tocopherol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">A17039</td>
			<td style="width:297px;">Antioxidant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Donkey serum</td>
			<td style="width:119px;">Bio-Rad</td>
			<td style="width:160px;">C06SBZ</td>
			<td style="width:297px;">Blocking reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glycine</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">3908.2</td>
			<td style="width:297px;">Background reduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Goat serum</td>
			<td style="width:119px;">Merck</td>
			<td style="width:160px;">S26-100ML</td>
			<td style="width:297px;">Blocking reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heparin sodium salt</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">7692.1</td>
			<td style="width:297px;">Background reduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hydrogen peroxide solution (30 %)</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">8070.2</td>
			<td style="width:297px;">Sample bleaching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methanol</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">4627.4</td>
			<td style="width:297px;">Sample pretreatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Paraformaldehyde</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">0335.3</td>
			<td style="width:297px;">Crystalline powder to make fixative solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium azide</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">K305.1</td>
			<td style="width:297px;">Prevention of microbial growth in stock solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">tert-Butanol</td>
			<td style="width:119px;">Alfa Aesar</td>
			<td style="width:160px;">33278</td>
			<td style="width:297px;">Sample dehydration for tissue clearing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">TO-PRO-3</td>
			<td style="width:119px;">Thermo Fisher</td>
			<td style="width:160px;">T3605</td>
			<td style="width:297px;">Nucleic acid stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triton X-100</td>
			<td style="width:119px;">Carl Roth</td>
			<td style="width:160px;">3051.2</td>
			<td style="width:297px;">Detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tween 20</td>
			<td style="width:119px;">AppliChem</td>
			<td style="width:160px;">A4974,0500</td>
			<td style="width:297px;">Detergent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Miscellaneous</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5 mL reaction tubes</td>
			<td style="width:119px;">Eppendorf</td>
			<td style="width:160px;">0030119401</td>
			<td style="width:297px;">Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Coverslip, circular (diameter: 22 mm)</td>
			<td style="width:119px;">Marienfeld</td>
			<td style="width:160px;">0111620</td>
			<td style="width:297px;">Part of imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Coverslip, circular (diameter: 30 mm)</td>
			<td style="width:119px;">Marienfeld</td>
			<td style="width:160px;">0111700</td>
			<td style="width:297px;">Part of imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Hypodermic needle (27 G x &#190;&#8221; [0.40 mm x 20 mm])</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:160px;">4657705</td>
			<td style="width:297px;">Filling of the imaging chamber with clearing solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">RTV-1 silicone rubber</td>
			<td style="width:119px;">Wacker</td>
			<td style="width:160px;">Elastosil E43</td>
			<td style="width:297px;">Adhesive for the assembly of the imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Ultimaker CPE 2.85 mm transparent</td>
			<td style="width:119px;">Ultimaker</td>
			<td style="width:160px;">8718836374869</td>
			<td style="width:297px;">Copolyester filament for 3D printer to print parts of the imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Technical equipment and software</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">3D printer</td>
			<td style="width:119px;">Ultimaker</td>
			<td style="width:160px;">Ultimaker 2+</td>
			<td style="width:297px;">Printing of imaging chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Automated water immersion system</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">15640019</td>
			<td style="width:297px;">Software-controlled water pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Benchtop orbital shaker</td>
			<td style="width:119px;">Elmi</td>
			<td style="width:160px;">DOS-20M</td>
			<td style="width:297px;">Sample incubation at room temperature (~ 150 rpm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Benchtop orbital shaker, heated</td>
			<td style="width:119px;">New Brunswick Scientific</td>
			<td style="width:160px;">G24 Environmental Shaker</td>
			<td style="width:297px;">Sample incubation at 37 &#176;C (~ 150 rpm)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Confocal laser scanning microscope</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">DMI 6000 TCS SP5</td>
			<td style="width:297px;">Inverted confocal microscope for sample imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Fiji</td>
			<td style="width:119px;">NIH (ImageJ)</td>
			<td style="width:160px;">open source software (v1.52h)</td>
			<td style="width:297px;">Image processing package based on ImageJ</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Long working distance water immersion objective</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">15506360</td>
			<td style="width:297px;">HC PL APO 40x/1.10 W motCORR CS2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vibratome</td>
			<td style="width:119px;">Leica</td>
			<td style="width:160px;">VT1200S</td>
			<td style="width:297px;">Sample slicing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:251px;">Workstation</td>
			<td style="width:119px;">Dell</td>
			<td style="width:160px;">Precision 7920</td>
			<td style="width:297px;">CPU: Intel Xeon Gold 5118 
			GPU: Nvidia Quadro P5000 
			RAM: 128 GB 2666 MHz DDR4 
			SSD: 2 TB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Primary antibodies</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:251px;">Goat anti-RABV N</td>
			<td style="width:119px;">Friedrich-Loeffler-Institut</td>
			<td style="width:160px;"></td>
			<td style="width:297px;">Monospecific polyclonal goat anti-RABV N serum, generated by goat immunization with baculovirus-expressed and His-tag-purified RABV nucleoprotein N 
			Dilution: 1:400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Rabbit anti-GFAP</td>
			<td style="width:119px;">Dako</td>
			<td style="width:160px;">Z0334</td>
			<td style="width:297px;">Polyclonal antibody (RRID:AB_10013382) 
			Dilution: 1:100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Rabbit anti-MAP2</td>
			<td style="width:119px;">Abcam</td>
			<td style="width:160px;">ab32454</td>
			<td style="width:297px;">Polyclonal antibody (RRID:AB_776174) 
			Dilution: 1:250</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:251px;">Rabbit anti-RABV P 160-5</td>
			<td style="width:119px;">Friedrich-Loeffler-Institut</td>
			<td style="width:160px;"></td>
			<td style="width:297px;">Monospecific polyclonal rabbit anti-RABV P serum, generated by rabbit immunization with baculovirus-expressed and His-tag-purified RABV phosphoprotein P (see reference 23: Orbanz et al., 2010) 
			Dilution: 1:1,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Secondary antibodies</td>
			<td style="width:119px;"></td>
			<td style="width:160px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-goat IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-mouse IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Donkey anti-rabbit IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Goat anti-mouse IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">Goat anti-rabbit IgG</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td style="width:160px;">depending on conjugated fluorophore</td>
			<td style="width:297px;">Highly cross-absorbed 
			Dilution: 1:500</td>
		</tr>
	</tbody>
</table>

high-resolution 3d imaging of rabies virus infection in solvent-cleared brain tissue

通过免疫标记对组织和器官的感染过程进行可视化是现代感染生物学的关键方法。观察和研究器官组织内病原体分布、病变和丰度的能力为疾病发展和进展提供了关键数据。使用传统的显微镜方法,免疫标签主要限于从石蜡嵌入或冷冻样品获得的薄部分。然而,这些薄截面的有限2D图像平面可能导致关于受感染器官复杂结构和感染细胞背景的关键信息丢失。现代多色、免疫染色兼容的组织清除技术现在提供了一种相对快速和廉价的方式来研究受病毒感染的器官组织的大容量 3D 图像堆栈。通过将组织暴露于有机溶剂中,它变得光学透明。这与样品的折射率相匹配,最终导致光散射的显著减少。因此,结合长距离自由工作距离目标,传统的共聚焦激光扫描显微镜(CLSM)可以以高分辨率成像高达1毫米的大组织部分。在这里,我们描述了一个协议,在组织清除后应用深层组织成像来可视化受感染大脑中的狂犬病病毒分布,以便研究病毒发病机制、传播、肌动和神经入侵等主题。

iDISCO14和 uDISCO13结合高分辨率 CLSM,可深入了解脑组织 RABV 感染和周围细胞环境的时空分辨率和可塑性。
使用RABV磷蛋白(P)的免疫染色,在小鼠大脑的厚部分可以可视化受感染神经元细胞的复杂层(图3)。随后,可以重建采集图像堆栈的无缝 3D 投影(图 3A、B、右侧面板;动画图 1?...

Watch this Scientific Journal Video about High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue at JoVE.com

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

新颖的免疫染色兼容组织清除技术,如溶剂清除器官的终极 3D 成像,允许狂犬病病毒脑感染及其复杂的细胞环境的 3D 可视化。厚厚的抗体标记脑组织切片在光学上透明,以增加成像深度,并通过共聚焦激光扫描显微镜实现 3D 分析。

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

该协议支持病毒感染的可视化，并提供对感染环境及其周围细胞环境的时空分辨率的深入见解。免疫污染的适用性不仅允许检测病毒，而且实际上也允许使用各自的细胞标记区分各种细胞亚组。将组织固定为免疫污点，将大脑样本在PBS中以4%的半成甲醛的1比10的比例放置，在4摄氏度下对组织体积进行至少48小时的采样。在固定期结束时，每次洗涤在 PBS 中洗三次组织样品至少 30 分钟，然后将样品在 4 摄氏度时将 PBS 中的 02%亚兹化钠转移到。然后使用振动器设置为 0.3 至 0.5 毫米/秒刀片进给速率，将组织分成 1 毫米厚的部分。将部分储存在PBS中，温度为4摄氏度。对于甲醇预处理将样品浸入上升的甲醇系列中，在室温下，每孵育一小时，轻轻振荡。最后一次孵育后，将样品冷却至摄氏四度。用四毫升预冷漂白溶液代替 100%甲醇，在摄氏四度下进行过夜孵化。第二天早上，用四毫升新鲜80%甲醇代替漂白溶液，在室温下孵育一小时。然后将样品浸入一系列降下降的甲醇中，与刚才演示的相反，在 20%甲醇浸入后，将样品在 4 毫升 PBS 中洗涤一小时。对于脑组织部分的免疫吸收，在PBS中洗涤样品两次，用4毫升0.2%Triton X-100洗涤样品，随后在4毫升37摄氏度的渗透，在0.2%Triton X-100、20%二甲基硫化物和0.3万酸二苯醚中渗透两天。在37摄氏度下，在PBS中用4毫升0.2%Triton X-100、10%二甲基硫化物和6%的正常血清阻断任何非特异性结合，为期两天。在阻断孵育标签结束时，样品用两毫升原抗体溶液在37摄氏度下5天，在2天和1~2天后刷新抗体溶液。接下来，在PBS中每毫升肝素的4毫升0.2%Tween 20和10微克中清洗样品一天，在一天中至少更换4至5次洗涤缓冲液，并在一夜之间留下最后一次洗涤。第二天，在2毫升二次抗体溶液中孵育样品，在37摄氏度下孵育5天。然后用四毫升的新鲜 0.2% Tween 20 和 10 微克每毫升肝素在 PBS 中清洗样品，如证明。用于样品的核染色，将样品孵育为核酸染色的四毫升 TO-PRO-3 中，保护样品五小时免受光。在孵育结束时，在PBS中每毫升肝素中洗涤样品0.2%Tween 20和10微克，随后在上升的tert-丁醇系列中脱水两小时。90%浸入后，将样品一夜之间转移到96%的三甲醇溶液中。第二天早上，将样品进一步脱水在100%t-butanol中两个小时，然后清除新鲜准备的BABB-D15中的组织部分两到六个小时，直到它们具有光学透明。然后，样品可以存储在 BABB-D15 中，防止光线，直到其安装和成像。对于样品安装，请使用 3D 打印机打印成像室和盖子。使用单组分室温硫化硅橡胶将直径 30 毫米的盖玻片安装到成像室上。同样，在盖子上安装 22 毫米盖玻片。使用水湿棉签去除两个环上的多余的硅橡胶，然后让橡胶通宵固化。第二天，在成像室中放置样品，在插入盖子之前，将一小体积的BABB-D15添加到成像室中。使用皮下针将腔内用 BABB-D15 填充腔室，并在用硅橡胶密封成像室之前将入口插入。要设置图像采集，请选择用于荧光灯的适当激光线，并调整每个探测器的检测范围，以防止信道之间的信号重叠。然后设置采集参数以查找 Z 堆栈的上边框和下边框并获取图像堆栈。要生成图像堆栈的 3D 投影，请打开斐济的图像文件，然后选择"图像颜色通道工具更多"和"分割通道"，将合并的图像拆分为各个通道。对于图像的漂白校正，对于每个通道，请选择"图像调整漂白校正"和"简单比率"。使用滑块调整每个通道的亮度和对比度，然后选择"图像颜色"和"合并通道"。勾选"创建合成"选项。将图像转换为 RGB 格式，并选择"图像堆栈"和"3D 项目"以生成 3D 投影。选择最亮点作为"投影"方法，并设置切片间距以匹配获取的图像堆栈的 Z 步进大小。为了达到最高质量，将旋转角度增量设置为 1 并启用插值。根据需要修改"总旋转"、透明度阈值和不透明度。然后将 3D 投影另存为 tiff 和 AVI 文件格式。利用狂犬病病毒磷蛋白复合层的受感染神经元细胞的免疫感染，可以在小鼠脑组织样本的厚部分中可视化。随后，可以重建采集图像堆栈的无缝 3D 投影。由于获得图像堆栈的高分辨率，感染可以评估到单个细胞级别，从而允许断言，例如，有关在感兴趣的细胞中抗原的丰度和分布。除了小鼠脑组织外，该协议还可以应用于其他动物物种的脑组织样本。例如，从受感染的雪铁龙大脑的不同隔间获得的部分显示不同程度的狂犬病病毒感染。星细胞可以通过胶质纤维酸蛋白的表达进行分化，而中性可以专门染色用于微管相关蛋白II. 同时，病毒蛋白可以共同染色，以评估受感染细胞与高光细胞亚群之间的关系。对于免疫保持抗体的选择，以检测感兴趣的蛋白质是至关重要的。虽然标准免疫体化学浓度通常是一个很好的起点，但一些抗体可能需要额外的调整。本协议中使用的许多化学品具有有害特性。穿着适当的个人防护装备（包括实验室外套和手套）在烟罩中进行相应的实验。

Research

JoVE Journal

Neuroscience

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

科研

神经科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

高分辨率人类中脑功能磁共振成像方法

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

大鼠脑组织被提取物的高分辨核磁共振代谢组学分析

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

高分辨率结构磁共振人类皮质下的成像<I&gt;在体内</I&gt;与死亡

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...