去除<em>果蝇</em从幼虫鱼片的感官神经元和表皮细胞的免疫荧光分析>肌肉组织
Summary
使用的果蝇幼虫树突分支（DA）神经元的神经元形态的研究通过免疫荧光在神经细胞和表皮蛋白质的原位可视化中受益。我们描述通过从幼虫体壁除去肌肉组织提高多巴胺神经元和包围的表皮细胞的免疫荧光分析的过程。
Abstract
的果蝇幼虫树突分支（DA）神经元是研究神经元形态发生机制流行模式。 DA能神经元发展与表皮细胞它们所支配，因而从免疫荧光两种neuronally和epidermally表达蛋白的原位可视化他们的分析好处沟通。制备用于免疫荧光实验幼虫鱼片的传统方法留下完好的肌肉组织，覆盖大部分的体壁，呈现几个挑战于成像神经元和表皮蛋白质。在这里，我们描述了从的果蝇幼虫鱼片去除肌肉组织的方法。该协议可允许否则由肌肉组织遮蔽蛋白成像，改进了信噪比，并方便了使用超分辨率显微镜来研究哒神经发育。
Introduction
果蝇幼虫树突分支（DA）神经元由于其顺从遗传操纵和与它们可以被成像的容易程度为研究神经元发育一个有价值的模型。这些感觉神经元已经在控制树枝状结晶形态1-3众多途径的鉴定工具。
四类DA能神经元（类别I – IV）支配幼虫表皮。这些神经元位于基底膜和表皮之间，与他们的树突形成基本上二维阵列4,5。四个班，IV级DA能神经元具有最高度支乔木，像其他动物的感觉神经元，这些乔木的制定需要从邻近组织的内在因素，以及线索，尤其是表皮，为他们的发展6-9 。
研究确定怎么这样神经元和额外的神经元的事实ORS通过免疫荧光检测原位蛋白表达的能力控制树突形态效益。幼虫的外表皮是穿不透的抗体，但这个障碍是很容易通过行之有效的夹层方法-10,11-制备幼虫鱼片克服。然而，正好位于内部到基底膜体壁肌肉组织呈现朝大神经元和表皮细胞可视若干挑战。首先，肌肉组织，这行最体壁，大大掩盖从神经元或表皮组织发出的荧光信号。这大大降低了信号与样品中的信噪比。第二，许多相关的蛋白质可以在肌肉组织中，以及在神经元或表皮来表示。此肌肉衍生的荧光信号可能从神经元或表皮荧光信号的进一步晦涩检测。最后，先进的显微技术不在亚衍射分辨率样品W的许可证成像并可能挑剔了在神经元中表达的蛋白质的定位和周围的表皮细胞12,13尤其有用。然而，通过从强信噪比和样品到盖玻片接近超高分辨率显微镜好处成像。除了降低信噪比，幼虫体壁肌肉的距离从盖玻片，从而限制可与超分辨率显微镜方法来实现改进的图像分辨率达的神经元。除了为免疫荧光分析挑战，肌肉组织提出从感觉神经元在幼虫体壁的障碍，电生理记录。因此，它有利于去除感觉神经元14的神经生理学操纵。
此处被描述为手动去除果蝇幼虫的肌肉组织的方法。我们证明了我们的协议p蛋白质ermits免疫荧光成像被另有肌肉组织遮蔽，提高了信号到第IV类多巴胺神经元的可视噪声比，并允许使用超分辨率显微镜，以更好地辨别在多巴胺神经元的蛋白质和细胞结构的空间关系和表皮。
Protocol
Representative Results
Discussion
在这里，一个协议是从的果蝇幼虫鱼片手工清除肌肉组织的描述。该协议会修改先前描述的幼虫解剖技术10,11。幼虫是在有机硅弹性体解剖的菜后，背中线的位置。一个单一的镊子尖头在其尽可能平坦的方向，仔细插入肌肉组织和表皮之间，靠近背中线。镊子轻轻向上从一个锚点中的每个感兴趣的幼虫段被拉至单独的肌肉组织。然后幼虫鱼片被固定在冷甲醛之后钳子再次用于从剩余…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢加里Laevsky对显微镜有益的讨论。这项工作是由美国国立卫生研究院资助的授予R01GM061107和R01GM067758到ERG
Materials
| Dumont #5 tweezers | Electron Microscopy Sciences | 72701-D | |
| Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips | World Precision Instruments | 14003 | |
| Sylgard 184 silicone elastomer kit | Dow Corning | 3097358-1004 | for dissecting plates |
| Austerlitz insect pins, 0.1 mm | Fine Science Tools | 26002-10 | |
| Fostec 8375 light source | Artisan Technology Group | 62792-4 | |
| Zeiss Stemi 2000 | Carl Zeiss Microscopy | ||
| Vectashield antifade mounting medium | Vector Laboratories | H-1000 | for confocal microscopy |
| Prolong Diamond antifade mountant | Life Technologies | P36970 | for structured illumination microscopy |
| Micro cover glass, 22×22 mm, No. 1.5 | VWR | 48366-227 | |
| Superfrost Plus microscope slides, 25 x 75 x 1.0 mm | Fisherbrand | 12-550-15 | |
| Mouse anti-Coracle antibody | Developmental Studies Hybridoma Bank | C615.16 | supernatant, dilute 1:50 |
| Mouse anti-Discs large antibody | Developmental Studies Hybridoma Bank | 4F3 | supernatant, dilute 1:50 |
| Rabbit anti-dsRed antibody | Clontech | 632496 | dilute 1:1000 |
| Goat anti-rabbit antibody, Alexa Fluor 568 conjugated | ThermoFisher Scientific | A-11011 | dilute 1:1000 |
| Goat anti-mouse antibody, Alexa Fluor 488 conjugated | ThermoFisher Scientific | A-11001 | dilute 1:500 |
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