髓鞘有机小脑片培养的制备及免疫染色
Summary
本文提出了一种利用免疫组织化学方法制备小鼠小脑和髓鞘染色的有机切片培养方法, 该方法适用于研究中枢神经系统髓鞘和再髓鞘的机制。
Abstract
在神经系统中, 髓鞘是由髓鞘胶质细胞产生的复杂的膜结构, 它使轴突产生, 并促进快速的导电性。髓鞘改变已被证明发生在各种神经疾病, 其中它与功能缺陷。在这里, 我们提供了一个由小鼠有机型小脑切片组成的体外模型的详细描述, 该模型可以在培养中保持数周, 并进一步标记为髓鞘可视化。
Introduction
神经元是高度极化的细胞, 它包括一个接收来自其环境的输入的长质树突隔间和一个轴突, 确保电脉冲的产生和传播到其他细胞。快速传播和及时提供信息对于神经系统的正常运作至关重要。在脊椎动物中, 它是由髓鞘促进, 这允许增加轴突传导速度1。髓鞘是由髓鞘胶质细胞 (即中枢神经系统中的少突胶质细胞) 和外周神经系统 (PNS) 中的雪旺细胞) 所产生的质膜压实层形成的一种特殊结构。在中枢神经系统和 PNS 中, 轴交相互作用推动了专门轴突域的形成: Ranvier 的节点及其周围域、anoco因为和 juxtaparode2。由髓鞘绝缘的轴突段, 或节间, 与 Ranvier 的节点交替, 对应于在电压门控钠通道 (Na v) 中丰富的未髓鞘小域 (nav)。Ranvier 节点上 nav通道的高浓度和快速激活使动作电位得以再生, 并与髓鞘的绝缘性能相结合, 确保了沿轴突3的神经冲动.
髓鞘胶质细胞除了在加速神经冲动传导速度方面的作用外, 还为轴突提供代谢支持, 保持其长期完整性, 并参与其生存4,5。此外, 近年来, 髓鞘在整个生命过程中都得到了动态的调节, 因此大概参与了各种神经系统功能的调节和可塑性。因此, 沿轴突调整髓鞘的分布、数量、长度和厚度可能是一种新颖的方法, 可以对各种网络6、7、8进行微调。因此, 髓鞘的进化习得是感觉、运动和认知功能的关键过程, 轴突和胶质细胞之间相互作用的扰动越来越被认为有助于发育或获得的神经疾病9。
髓鞘成分的特点, 其特点是脂质比例高 (70%)与蛋白质相比 (30%)与其他细胞膜相比,10。然而, 与髓鞘脂质不同的是, 髓鞘蛋白大多是髓鞘特异性的, 包括髓鞘碱性蛋白 (MBP)、蛋白脂蛋白 (PLP)、2 ‘、3 ‘-环核苷酸 3 ‘-磷酸二酯酶 (CNP)、髓碱相关糖蛋白 (MAG),髓鞘少突胶质细胞糖蛋白 (MOG), PMP-22 和 P010.各种染色髓鞘的组织学方法, 如卢克索快速蓝11, 苏丹黑色 b12, 贝克酸血红素法13, 以及银染色14。然而, 这些方法并不总是允许足够的对比和分辨率来可视化单个纤维。检测髓鞘的另一种方法是针对髓鞘蛋白的免疫组织化学。各种抗体针对的是具有高度特异性的髓系特异性抗原, 可用于常规检测髓鞘结构。抗体-抗原相互作用可以进一步揭示使用二级抗体结合到荧光团针对的原代抗体和可视化与适当的荧光显微镜。在这里, 我们描述了一个免疫化学协议, 染色髓鞘在体内小脑切片上, 一个模型, 允许一个很好地保存神经组织结构。此外, Purkinje 细胞 (小脑的唯一髓鞘神经元) 的组织和大小使它们成为电生理研究的经典模型, 它们同样是进行固定或活体成像研究的理想选择。
小脑切片由 P9-p10 小鼠生成, 这一时间与 Purkinje 细胞髓鞘的早期发生相对应, 这一过程主要是通过体内一周 (体外 6-7, DIV)15来实现的。此外, 该模型适用于研究脱髓鞘疾病, 如多发性硬化症 (MS), 因为广泛脱髓鞘可以诱导小脑片使用骨髓毒性化合物溶磷酸胆碱 (或溶菌素, LPC),随后是 16,17 的自发再髓鞘。内源性再髓鞘发生在 LPC 从培养基中取出后的两天内膜再髓鞘, 几乎在治疗一周后完成。
完成该协议大约需要3周的时间, 包括半天的小脑切片培养准备, 一周获得完全髓鞘切片, 然后2天达到脱髓鞘的高峰, 再过一周的时间进行充分的准备。再髓鞘。此外, 免疫组织化学可在2天内完成。这里描述的协议适用于6只小狗的标准垃圾, 需要根据计划中的实验所使用的动物数量进行调整。
Protocol
Representative Results
Discussion
在这里, 我们详细介绍了一个方案, 以产生一个与小鼠小脑有机切片培养相对应的体外模型, 该模型改编自先前公布的方法15、16、19和随后的髓鞘这些制剂的免疫染色。该策略提供了在健康和病理状态下使用高分辨率可视化髓鞘成分的可能性。
从10天大的小鼠身上提取的小脑有机切片培养是一个成熟的实验?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢肖恩·弗里曼博士、Nathalie Sol-Foulon 博士和 Thomas Roux 博士对手稿的宝贵评论。这项工作由 INSERT、ICM、ARSEP 赠款 R13123DD、ANR R17127DD (至 a. d.) 和 FRM 研究金、SPF20110421435 (至 a. d.)、FDT201-704332 (至 m. t.) 提供资金。我们感谢 CELIS 细胞培养设施和冰毒。Quant 成像平台。
Materials
| BME medium | ThermoFisher Scientific | 41010026 | |
| Hank’s Balanced Salt Solution (10X HBSS) | ThermoFisher Scientific | 14180046 | |
| GlutaMAX (100X) | ThermoFisher Scientific | 35050038 | |
| Heat-inactivated Horse Serum | ThermoFisher Scientific | 26050088 | |
| Penicillin–Streptomycin (10.000 IU/mL) | ThermoFisher Scientific | 15140122 | |
| Gey’s Balanced Salt Solution | Sigma Aldrich | G9779-500ML | |
| D-Glucose Solution (45%) | Sigma Aldrich | G8769-100ML | |
| Lysophosphatidylcholine (LPC) | Sigma Aldrich | L4129-100MG | |
| Paraformaldehyde (PFA) | Electron Microscopy Sciences | 15714 | |
| Absolute ethanol (100% ethanol) | VWR Chemicals | 20821.330 | Cooled at -20°C |
| Triton® X-100 | Sigma Aldrich | X100-500ML | |
| 10% Normal Goat Serum (NGS) | ThermoFisher Scientific | 500622 | |
| Phosphate Buffer Solution | EuroMEDEX | ET330-A | pH 7.4 |
| Anti-GFP Antibody (Polyclonal, Chicken) | Merck Millipore | 06-896 | Dilution 1/300 |
| Anti-Myelin Basic Protein (MBP) Antibody (Polyclonal, Chicken) | Merck Millipore | AB9348 | Dilution 1/150 |
| Anti-Myelin Basic Protein (MBP) Antibody (Monoclonal, Mouse IgG2b) | Merck Millipore | NE1019 | Dilution 1/200 |
| Anti-PLP Antibody (Rat, Hybridoma) | Gift from Dr. K. Ikenaka; Okasaki, Japan | Dilution 1/5 to 1/10 | |
| Anti-Sodium Channel, Pan Antibody (Monoclonal, Mouse IgG1, clone K58/35) | Sigma Aldrich | S8809 | Dilution 1/150 |
| Anti-Caspr Antibody (Polyclonal, Rabbit) | Abcam | ab34151 | Dilution 1/500 |
| Goat Secondary Antibodies conjugated to Alexa Fluor 488, 594, 647 or 405 | ThermoFisher Scientific | Dilution 1/500 | |
| Fluoromount | SouthernBiotech | 0100-20 | |
| Tissue chopper | McIlwain | ||
| Razor blades | |||
| Large scissors | F.S.T | 14101-14 | |
| Small scissors | F.S.T | 91500-09 | |
| Fine-straight forceps | F.S.T | 91150-20 | |
| Curved-fine forceps | F.S.T | 11297-00 | |
| Cell culture dishes (60-mm and 100-mm) | TPP | ||
| 4-, 6-well culture plates | TPP | ||
| Millicell culture inserts (0,4 µm, 30-mm diameter) | Merck Millipore | PICM0RG50 | |
| Cell culture incubator | 37°C, 5% CO2 | ||
| Fine-end pipette tips | Dutscher | 134000CL | |
| Wide-bore pipette tips | ThermoFisher Scientific | 2079G | |
| Sterile syringe | Terumo Europe | 20 or 50 mL | |
| Sterile syringe filters | Terumo Europe | 0.22 µm | |
| Scalpel | Swann-Morton | 0510 | |
| Brush | |||
| Microscope slides | RS France | 76 x 26 x 1.1 mm | |
| Glass coverslips | RS France | 22 x 22 mm | |
| Kimtech Sciences Tissue Wipers | Kimberly-Clark Professional | 5511 | |
| Binocular microscope |
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