Name: removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells
Uploaded: 2016-11-02T13:00:00
Description: Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

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

注意：对于肌肉切除（ 图1）的步骤的制备幼虫鱼片先前描述的方法的变形例。之前和之后的肌肉除去步骤概述简要地并且读者可以参考以前的工作10，11，用于更详细的描述。 1.解剖幼虫在冷盐水<ol><li>准备冷HL3.1生理盐水15或冷的Ca 2+ HL3.1生理盐水11（ 表1）的工作稀释。放置幼虫与刚好足够冷盐水硅氧烷弹性体盘，...

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Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+bantam+regulates+a+developmental+transition+in+epithelial+cells+that+restricts+sensory+dendrite+growth.">The microRNA bantam regulates a developmental transition in epithelial cells that restricts sensory dendrite growth.</a> Development. 141 (13), 2657-2668 (2014).</li><li>Meltzer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermis-derived+Semaphorin+promotes+dendrite+self-avoidance+by+regulating+dendrite-substrate+adhesion+in+Drosophila+sensory+neurons.">Epidermis-derived Semaphorin promotes dendrite self-avoidance by regulating dendrite-substrate adhesion in Drosophila sensory neurons.</a> Neuron. 89 (4), 741-755 (2016).</li><li>Han, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+cells+are+the+primary+phagocytes+in+the+fragmentation+and+clearance+of+degenerating+dendrites+in+Drosophila.">Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila.</a> Neuron. 81 (3), 544-560 (2014).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Karim, M. R., Moore, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+Drosophila+larval+peripheral+sensory+neuron+dendrites+and+axons+using+genetic+mosaics.">Morphological analysis of Drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics.</a> J Vis Exp. 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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...

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。

<table border="0" cellpadding="0" cellspacing="0" width="1299">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dumont #5 tweezers</td>
			<td style="width:304px;">Electron Microscopy Sciences</td>
			<td style="width:151px;">72701-D</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td>
			<td>World Precision Instruments</td>
			<td>14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>for dissecting plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Austerlitz insect pins, 0.1 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fostec 8375 light source</td>
			<td>Artisan Technology Group</td>
			<td>62792-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss Stemi 2000</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Diamond antifade mountant</td>
			<td>Life Technologies</td>
			<td>P36970</td>
			<td>for structured illumination microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro cover glass, 22x22 mm, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Coracle antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>C615.16</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-dsRed antibody</td>
			<td>Clontech</td>
			<td>632496</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11011</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>dilute 1:500</td>
		</tr>
	</tbody>
</table>

removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

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。

我们证明肌肉切除过程的效用改善信噪比在免疫荧光实验共同形象化隔连接蛋白小艇（科拉）和光盘大型（DLG）用标记与膜标记IV级多巴胺神经元一起CD4- tdTomato。 科拉先前已用于识别阿凡达的神经元树突是由表皮细胞封闭，是已在研究协会DA神经元的形态和功能的树突4,6-9,16许多鉴定表皮因素之一大片。用抗DsRed的免疫染色，以检测神经元（红...

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

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。

去除<em&gt;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&gt;肌肉组织

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Research

JoVE Journal

Neuroscience

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

Functional Analysis of the Larval Feeding Circuit in Drosophila

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

从美国牛蛙的小囊（毛细胞的生理准备<em&gt;牛蛙</em&gt;）

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

跟踪果蝇幼虫行为对嗅神经元 Optogenetic 刺激的反应

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...