Name: paraffin-embedded and frozen sections of drosophila adult muscles
Uploaded: 2010-12-27T17:30:00
Duration: 7 min 28 s
Description: Watch this Scientific Journal Video about Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles at JoVE.com

我们感谢Eichele教授允许我们使用冷冻切片机。工作是由马克斯 - 普朗克Gesselschaft。

1。制备<ol><li>相结合无水乙醇，氯仿和冰醋酸的比例为6:3:1，刚准备卡诺的固定液。3本，以及所有淹没衣领应保持在玻璃染色罐（见试剂的建议部分）解决方案。 </li><li>准备铝箔口袋里的正确的衣领大小。 </li><li>染色罐准备以下解决方案：2 × 40％乙醇，70％的乙醇，2个100％的乙醇，甲基（MB），50/50 V / V MB和石蜡，石蜡2 ×。放置在一个孵化器设置的MB和石蜡的容器至60-65 ° C。 </li><li>暖石蜡倒入铝箔口袋60-65 ° C。 </li></ol> 2。在颈圈固定苍蝇<ol><li>连接下使用磁带双眼在一个垂直的位置，在那里你可以看到飞的切入点领4。 </li><li>麻醉苍蝇使用二氧化碳，或通过使用冰块的低温。要小心，不要冻结苍蝇。 </li><li>使用镊子，拿起抓住正确导向（刀片和腹部下方的叶片顶端的头部和胸部）到领他们的翅膀和地方的个别苍蝇。 10-20苍蝇应该很容易适应的衣领。 </li></ol>注意：如果你是几个基因型分析，不要忘记注意领口的号码和相应的基因型。 3。 果蝇 Thoraxes石蜡切片<ol><li>迁移的衣领，卡诺的解决方案，并组织定于4 °，在夜间彗星。 </li><li>固定，脱水后的样品，使用乙醇浓度的增加。 10分钟，每到40％（2次），70％和100％（2次）在室温下乙醇淹没的衣领。在MB和MB +石蜡溶液（1:1）下孵育领各30分钟，然后渗透在两个转变石蜡，每次60分钟，在60-65 ° C。中的领迅速搬迁到铝箔的口袋，领口和填写融化（60-65℃）石蜡。在室温下放置，并允许石蜡成为硬盘（最好是离开过夜）。请注意领子可放在在铝箔的口袋里，在不同的方向，取决于你需要的部分（纵向或横向）的方向。 </li><li>不折干蜡块用的铝箔的衣领，轻轻地从蜡块单独的领子。使用锋利的刀片或手术刀的帮助下，小心地切出多余的石蜡，从飞周围组织。 </li><li> 7-10微米节中的步骤，一个旋转切片机上切蜡块，让切组织漂浮在37℃水浴持平。极地幻灯片上放置切片组织，并允许在夜间干燥。这些幻灯片可以使用hematoxyline和曙红染色（图1A - D），苯胺蓝或甲苯胺蓝，其他死亡可视化的组织结构，以及抗体染色（图1E - E“）。 </li></ol> 4。冰冻切片的果蝇 Thoraxes <ol><li>与石蜡切片，准备在白领和时尚铝箔袋苍蝇。冻结冷却器将需要准备的样品。确保冷却器大约是-60 ° C时，用一个小的乙醇和干冰，以便达到这一温度。在实验开始前一小时在4℃冰箱冷冻包埋剂瓶（组织TEK华侨城复合）为了冷却下来，并尽量减少气泡的形成倒挂。 </li><li>迁移已预冷却几分钟内冻结冷却器和迅速填补与低温嵌入复合铝箔口袋苍蝇的衣领。让冻结的样品为3-10分钟。小心地解开冷却器内形成块，轻轻分开嵌入块的衣领，并至少有一天它在-20 ° C。 </li><li>在冷冻切片机-15和-18℃之间的冷冻肌肉切与切片厚度为10-15微米。放置在两极化的幻灯片，并保持在-20 ° C，直到准备做进一步的处理。我们建议在4％多聚甲醛PBS液固定抗体染色前在室温下10分钟的组织。 </li></ol> 5。在果蝇肌肉的血脂检测脂滴可使用Sieber和Thummel 5通过了一项协议冰冻切片油红O染色检测。 <ol><li>固定后，用清水洗净幻灯片两次5分钟，10分钟和3 h油红O染色在室温下孵育在丙烯乙二醇平衡。然后洗净样品丙二醇和30分钟的PBS的5分钟的2倍。安装在30％甘油。 </li></ol> 6。代表性的成果： <img src="/files/ftp_upload/2438/2438fig1.jpg" alt="图1" /> 图URE 1。 Parrafin -包埋切片 Hematoxyline和曙红染色横（AB）和纵向（CD）第间接飞行肌。 A和C显示正常结构的肌肉。由大小和形态的肌肉异常，表示在B和D分别为（黑色箭头）。 果蝇胸部染色与反LamC，核膜标记和DAPI染色，核染色（英）的横截面。放大的正常（红色箭头）和恶化（黄色箭头）肌肉（F）的。 G代表LamC和DAPI 果蝇肠道染节。 <img src="/files/ftp_upload/2438/2438fig2.jpg" alt="图2" /> 图2。冰冻切片 A.横向冰冻切片油红O，脂滴标签果蝇与胸部沾满。 B.间接飞行肌纵冰冻切片与抗- DG，肌肉肌膜标记和DAPI染色。

<ol>
	<li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+anatomy+and+histology+of+the+imago+of+Drosophila+melanogaster.">The internal anatomy and histology of the imago of Drosophila melanogaster.</a> , (1950).</li><li>Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+muscle+and+neuronal+disorders+in+a+Drosophila+model+of+muscular+dystrophy.">Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy.</a> The EMBO journal. 26, 481-481 (2007).</li><li>Kucherenko, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modifier+screens+reveal+new+components+that+interact+with+the+Drosophila+dystroglycan-dystrophin+complex.">Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.</a> PloS one. 3, e2418-e2418 (2008).</li><li>Puchtler, H., Waldrop, F. S., Conner, H. M., Terry, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnoy+fixation:+practical+and+theoretical+considerations.">Carnoy fixation: practical and theoretical considerations.</a> Histochemie. 16, 361-36 (1968).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila - A Laboratory Manual. , (1989).</li><li>Sieber, M. H., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DHR96+nuclear+receptor+controls+triacylglycerol+homeostasis+in+Drosophila.">The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila.</a> Cell metabolism. 10, 481-481 (2009).</li></ol>

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stainless steel collars</td>
<td>&nbsp;</td>
<td>self-made</td>
<td>&nbsp;</td>
<td>Specially constructed</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools GmbH</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Blade or scalpel</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wheaton macro staining jar</td>
<td>&nbsp;</td>
<td>Wheaton Science Products </td>
<td>900200</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>&nbsp;</td>
<td>Model: Hyrax M25</td>
</tr>
<tr>
<td>Cryo-microtome</td>
<td>&nbsp;</td>
<td>Leica </td>
<td>&nbsp;</td>
<td>Model CM3050S</td>
</tr>
<tr>
<td>60-65&deg;C Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>Large enough to hold at least 4 staining jars</td>
</tr>
<tr>
<td>Freezing cooler with metal block</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>Store at -80&deg;C</td>
</tr>
<tr>
<td>Super-frost slides</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>9161155</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Any </td>
<td>&nbsp;</td>
<td>Recommend 24 X 40 mm</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>288306</td>
<td>Analytical grade </td>
</tr>
<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>100063</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>100983</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Methylbenzoate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M29908-500G</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Paraplast plus</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>76258</td>
<td>Paraffin</td>
</tr>
<tr>
<td>Tissue-Teck O.C.T. compound</td>
<td>&nbsp;</td>
<td>Sakura</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>16% formaldehyde, methanol free</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>18814</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G5150-1L</td>
<td>&nbsp;</td>		</tbody>
		</table>
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paraffin-embedded and frozen sections of drosophila adult muscles

在人类肌营养不良症和肌病的分子特性，揭示了肌肉规范的肌肉疾病和遗传分析的复杂性，在模型系统的形成和功能已经转化为肌肉生理学提供了有价值的见解。因此，识别和表征背后肌肉损伤的分子机制是至关重要的。成年果蝇多纤维肌肉的结构类似于脊椎动物横纹肌1 和果蝇的基因可追踪已是一个很大的系统来分析营养不良肌肉的形态和特征的进程，影响肌肉功能老化成年苍蝇2。在这里，我们现在准备石蜡包埋，并冻结了部分果蝇胸肌肉组织学技术。这些准备工作要与古典组织的污迹，并与蛋白质检测染料染色标记的组织，允许和专门的冰冻切片是在完整的肌肉蛋白的免疫组化检测的理想选择。这使得肌肉组织结构，形态缺陷的鉴定，并检测肌肉/果蝇成人肌肉中的神经元特异性蛋白质的表达模式分析。这些技术也可以用于身体其他部位的切片略作修改。

Watch this Scientific Journal Video about Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles at JoVE.com

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

鉴定肌肉损伤的基本机制是至关重要的。在这里，我们现在准备石蜡包埋，并冻结了部分果蝇胸肌肉组织学技术。这使得肌肉的形态和本地化的蛋白质和其他肌肉细胞成分分析。

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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Biology

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Whole Cell Recordings from Brain of Adult Drosophila

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High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

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Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

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The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Oenocytes from Adult Drosophila melanogaster

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In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

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Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

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Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

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Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

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Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...