我们诚挚地感谢萨宾Reckinger优秀的技术援助和支持。

1.准备<ol><li> 样品采集 注意:可用于单个或合并肌肉样品的磁性搅拌器方法。 <ol><li>从适当的好发部位，并以适当量9收集样品。请参阅该国要求或信息和通信技术，世界动物卫生组织或ISO的建议。 <ol><li>对于欧盟，例如，采取从隔膜支柱1g的样品在过渡到国内的猪的强壮有力的一部分。确保所有的肌肉样本不含所有的脂肪和筋膜。 </li></ol></li><li>如果舌头测试，删除之前测试的表面层。 </li><li>请注意，如果样品被冻结，因为大多数旋毛虫不抗冻和死亡后解开，是消化不太耐倾向于坚持容器壁，从而降低检测灵敏度。 注意:如果冻结，加倍为L样本量东和增加沉降时间（见下文）。 </li></ol></li><li> 消化液的制备 <ol><li>添加25％的盐酸（16±0.5毫升），以2升46预热自来水 - 48℃到3升的玻璃烧杯中。过低的温度会导致消化不完全;过高的温度停用胃蛋白酶的酶学性质。 </li><li>放置一个搅拌棒的烧杯中，并搅拌一预热板溶液（46 - 48℃）。 </li><li>添加10±0.2克胃蛋白酶（1:10,000 NF，1:12500 BP公司，2000 FIP）到酸性溶液中。 注意:胃蛋白酶的质量是最重要的和酶的活性必须由生产者进行认证。对于实验室安全原因，并保持胃蛋白酶活性，增加消化液的组件的顺序必须坚持。 </li></ol></li><li> 肌肉样品制备 <ol><li>砍在搅拌机或粉碎机百克肌肉样本。为了方便BLEN丁，加2毫升预热水样品和以最大速度融合为2 - 3秒。 </li><li>第二混合前，打开搅拌机和混合碗缘缘的底部，并重复交融的顶级传送任何肌肉样本。继续以2连发混合 - 3秒，直到肉没有明显的碎片依然存在。 注:太少混合，可能会导致消化不完全，而过多的混合可以在样品6损坏旋毛虫存在。 </li></ol></li></ol> 2.程序<ol><li> 肌肉样本消化 <ol><li>传送1升消化流体成圆筒。转移绞肉放入烧杯中，并在消化流体用勺子或叉子传播。彻底用500mL的预热消化流体进3升烧杯中漂洗搅拌器盖子，叶片和搅拌器碗。 注:可导致SENS的损失不完全漂洗itivity。 </li><li>用铝箔覆盖烧杯，并保持44一恒定温度 - 46℃，用一温度计监测。 </li><li>炒出的消化液30分钟，无飞溅创建了深刻的漩涡，直到肉颗粒消失。根据测试肉（野生动物如肉）的类型，增加消化时间直至最多60分钟。 </li></ol></li><li> 过滤和消化流体的沉降 <ol><li>以确定未消化的组织的量，调整刻度为零，在使用前称量筛，并注意其重量。筛应清洁，没有任何组织的粒子，这可能阻碍旋毛虫到达沉降漏斗。检查分液漏斗垂直平整。 </li><li>消化后，小心地倒入通过180微米筛消化液到分离玻璃漏斗。分离玻璃漏斗的宽度不应bË长度大于55％，使良好的幼虫沉淀。请注意，以避免任何溢出。 </li><li>为了避免灵敏度降低，彻底冲洗玻璃烧杯中，并从一个挤压洗涤瓶用约200毫升自来水的筛和漂洗水转移到沉淀玻璃漏斗。小心仔细冲洗3升烧杯中的边缘。提起筛子，彻底筛下冲洗的面积。 </li></ol></li><li> 未消化的组织量的确定 注意:由于在该方法的执行错误，肌肉组织可以保持在筛上，以及筋膜和结缔组织这是难消化。未消化的组织的量在第一10分钟沉淀步骤（见2.5）来测定。这允许大约30分钟的筛干，残留水会影响未消化的组织所确定的权重。 <ol><li>广场上规模纸巾，并权衡与undiges筛特德组织。 </li><li>从未消化组织筛子过滤重量减去前筛的重量。如果起始样品重量的不超过5％保留在筛（ 即 5克/ 100克原料）在消化过程被认为是令人满意的。 </li><li>如果未消化的组织的量超过5％时，重复使用新鲜肌肉样品的测试。如果剩余的组织主要是由未消化的肌肉组织的和新鲜样品不可用，再次消化未消化遗体和除了原始样品检查。 </li></ol></li><li> 消化液的沉淀 <ol><li>保持过滤消化流体在分液漏斗中30分钟。如果不受干扰，在旋毛虫会沉淀在漏斗的底部。 </li><li>如果使用冷冻的样品，增加沉降时间为最多60分钟。 </li><li>一旦沉积完成后，完全打开活塞分液漏斗中，让至少40消化液毫升迅速跑出来，放入量筒（80毫升，如果肌肉样品已经先前冻结）。 </li><li>首先，让一半的流体的进入气缸，然后在相反的方向完全打开活塞，以允许10毫升消化流体的通过。最后，在另外10毫升相反的方向打开活塞。此过程可确保旋毛虫不会被困在水龙头。 注意:需要足够的速度，以避免残留在分液漏斗中的幼虫，降低灵敏度。 </li></ol></li><li> 气缸中的消化流体的沉降 <ol><li>离开气缸中的沉淀10分钟，以允许幼虫沉积物。通过用移液管抽吸，而不会干扰10mL的沉积物除去30毫升上清液。 </li><li>以减少样品中的组织的纤维的量，30毫升的自来水中添加的沉积物和离开另一个10米英寸重复，直到液体是明确的。 </li><li>除去上清液，转移至10毫升洗净泥沙，以网格化培养皿或幼虫计数盆地。冲洗用10mL自来水筒，然后将水添加到样品中。 注意:在样品中作为大量碎片能够防止旋毛虫 （ 图1）的标识，如果需要的话应进行进一步的洗涤步骤，。 </li></ol></li><li> 镜检 <ol><li>使用trichinoscope或立体显微镜具有可调强度的子级透射光源在一个15至20倍的放大倍率的洗涤步骤后，立即以检查样品。 </li><li>浇样品放入网格培养皿后，检查由电网盘/盆系统网格。如果没有发现犯罪嫌疑人的结构，轻轻移动在网格培养皿或幼虫计数盆地流体以循环的方式让任何旋毛虫拉rvae，这潜在地忽略，在培养皿的中心积聚。重复检查。 </li><li>如果发现可疑的结构，提高放大倍数40 - 100X。从盘/盆用移液器除去旋毛虫 ，并与90％的乙醇的管收集。 </li><li>完整的菜已经被审查后，重复上述步骤，从盘/盆的轻轻摇动开始。重复该过程至少三次或直到没有更多的幼虫被找到。 </li><li>计数幼虫的数目。关联到肌肉组织的量测试和本为每克（LPG）的幼虫。 </li><li>如果太多的幼虫存在于消化流体以确定幼虫数可靠地，返回包含幼虫量筒中的流体，从挤压洗瓶用自来水冲洗，并留下幼虫沉降至底部。 </li><li>一个10分钟沉降时间后，降低上清液到规定体积（ 例如10毫升）中。以确定确切体积，转移上清液到新的筒用吸管。 </li><li>通过剧烈摇晃液均匀暂停幼虫。在摇动运动，加入20微升悬浮幼虫12滴到培养皿。 </li><li>检查显微镜每下降。确定在240微升的幼虫数和删除的最高和最低计数。推断幼虫的数目，以上清液的总体积（ 例如 ，10毫升）。 注意:立体显微镜的质量是最重要的为旋毛虫的识别。 </li><li>当幼虫在管通过吸移管收集，仔细检查幼虫不粘附在移液管壁而被转移到管中。 注:从合并的样品阳性结果旋毛虫必须跟踪从池中回原籍的尸体。在这里，池大小应逐步减少直至正面胎体鉴定者tified。样本大小也可以在这个过程中增加以增加灵敏度。其中集体样品的检查产生一个正的或不确定的结果，另外20克样品从每个猪服用。从五头猪各20 g样品汇集并使用所描述的方法检测。以这种方式，从20组的五头猪样本将被检查。当一个合并样品中的五头猪检测旋毛虫 ，将20g样品从该组中的个别猪收集并直到检测原点的正胎体每个单独检查。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

虽然磁力搅拌器方法可以容易地进行，而不是严格遵守的技术细节导致灵敏度不足，从而危及消费者的健康。关键控制点已经凸显在上面的文字。此外，使用适当的设备是为测试的成功结果至关重要。所有船只应作出的涂有硅，以减少对表面幼虫附着玻璃和枪头。 消化方法的灵敏度取决于在肌肉幼虫密度和肌肉样品的测试的量。为3的幼虫密度- 5液化石油气肌肉组织的，100％的灵敏度被报道，但低于1 LPG的灵敏度下降至40％，13。取决于所测试的宿主动物种类，试验的灵敏度可通过增加样品的使用量得到改善。第i在野生动物nfection负担通常较低，因此大尺寸的样品使用14。好发部位也宿主动物的不同而不同。这里，一些肌肉组织的下消化率如舌需要被考虑在内，这也可导致更大的样本在尺寸15。 此外，要明白，磁力搅拌器方法不包括内部控制是重要的。因此，从采样质量保证管理的文档是必不可少的，以获得准确的和精确的结果。实验室性能检测旋毛虫肌肉样品中的质量应在能力测试每个分析师经常参与进行监测。 该方法的进一步限制是由显微镜肌幼虫的最终识别阶段是高度主观和HEAvily依赖于幼虫形态的研究分析师的知识。 绕过这个问题的一个有趣的替代方法是磁力搅拌器方法/过滤器上分离之后幼虫检测通过胶乳凝集试验。不过，根据欧盟法规此方法仅视为等同于家猪的而不是动物，它们在感染风险较高的肉类的测试，如野猪4。用单克隆抗体幼虫抗原的识别使得测试更加客观，但否认实验室确定每克肉16的幼虫数的可能性。 磁力搅拌器方法的进一步变化包括机械辅助合并样本消化法/沉淀技术，机械辅助合并样本挖estion方法/过滤器上分离的技术，并为最多至35g技术的合并样品的自动消化方法。这些技术都是基于肉和旋毛虫的可视化及数量的人工消化，但在用于过滤和沉降步骤的设备不同。 分离出的幼虫可以进一步确定在通过分子测试（ 如多重PCR）17种的水平。根据欧盟法律，所有的阳性样品必须转发国家参考实验室或EURLP为旋毛虫决心。

属旋毛虫寄生线虫可在全球范围内的食肉动物和杂食的哺乳动物，鸟类和爬行动物的横纹肌进行检测。这些人畜共患线虫可以从猪，马及人的时候生的或半生肉和肉类衍生产品，以及野生动物摄入。此人畜共患可以是在人类特征在于特异病征性体征和症状， 例如腹泻，发烧，眶周水肿和肌痛和可能的并发症，如心肌炎，血栓栓塞性疾病和脑炎1一种严重的疾病。 旋毛虫是野生和家养动物旋毛虫感染的最普遍的病原体，导致大部分人类感染全世界。在欧洲，北非和西非，和西亚， 旋毛虫britovi是人类感染的另一个来源。此外，另外十个类群较少通常报告为致病<html>人类疾病和剂在世界的不同地区被发现，通常在野生动物2。除了野生动物如野猪，熊，海象和獾，猪代表了人类感染的全球最重要的来源。 为防止旋毛虫病的最好方法就是从吃生肉产品不要和煮任何肉类，尤其是野味到安全温度（&gt; 65℃1分钟， 即从粉红肉的颜色变成棕色的核心肉产品）3。欧盟和美国农业部已指定冷冻和烹调时间和温度，可以用来杀死旋毛虫幼虫肉4,5猪肉制品。此外，对于旋毛虫在肉及肉制品灭活建议由国际委员会发表的旋毛虫病"外部参照"&gt; 6。在欧洲，除了引进的商业猪群那里旋毛虫感染的风险可以忽略不计控制住房条件，人类旋毛虫病的预防是基于易感动物4肌肉样品的实验室检查。 对于食品安全而言，消化试验是在肉3,7直接检测旋毛虫的唯一可靠方法。即使有消化测定的几个变型中，磁力搅拌器的方法是国际公认的参考方法3，4，7。该测定是基于在横纹肌组织的检测旋毛虫 。肌肉样品的酶消化和随后的过滤和沉淀步骤， 三棱后chinella幼虫通过显微镜鉴定。根据贸易义务和国家立法，也有磁力搅拌器方法的一般协议的小变化，众说纷纭。在这里，技术细节是根据欧盟要求的4，ISO规格8，信息和通信技术指导6，和欧盟参考实验室的寄生虫（EURLP）的经验和德国的参考实验室旋毛虫 。

<table><tbody><tr><td>Knife, Scissors, Tweezers</td><td></td><td></td><td>Cutting samples and removing non-digestible tissue</td></tr><tr><td>Blender&amp;nbsp;</td><td></td><td></td><td>With a sharp chopping blade</td></tr><tr><td>Magnetic stirrer</td><td></td><td></td><td>With thermostatically controlled heating plate</td></tr><tr><td>Stirring Rod</td><td></td><td></td><td>Teflon-coated, approximately 5 cm long</td></tr><tr><td>Conical glass separation funnel</td><td></td><td></td><td>Capacity of at least 2 L, preferably fitted with Teflon safety plugs. The width should not be larger than 55% of the length to allow a good larva sedimentation.</td></tr><tr><td>Stands, rings and clamps</td><td></td><td></td><td></td></tr><tr><td>Sieve</td><td></td><td></td><td>Mesh size 180 microns, external diameter 11 cm, with stainless steel mesh</td></tr><tr><td>Funnel</td><td></td><td></td><td>Internal diameter not less than 12&amp;nbsp;cm, to support the sieves</td></tr><tr><td>Glass beaker</td><td></td><td></td><td>Capacity 3 L</td></tr><tr><td>Glass measuring cylinder</td><td></td><td></td><td>Capacity 50 to 100 mL, or centrifuge tube</td></tr><tr><td>Stereo-microscope</td><td></td><td></td><td>With a substage transmitted light source of adjustable intensity or trichinoscope with a horizontal table</td></tr><tr><td>Petri dishes</td><td></td><td></td><td>9 cm diameter, marked on their undersides into 10 &amp;times; 10 mm square examination areas using a pointed instrument or a larval counting basin for the tricinoscope</td></tr><tr><td>Aluminium foil</td><td></td><td></td><td>Alternatively a lid to cover the beakers</td></tr><tr><td>25% hydrochloric acid</td><td></td><td></td><td></td></tr><tr><td>Pepsin</td><td></td><td></td><td>Strength: 1:10,000 NF (US National Formulary) corresponding to 1:12,500 BP (British Pharmacopoeia) and to 2,000 FIP (F&amp;eacute;d&amp;eacute;ration internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/mL</td></tr><tr><td>Ethanol</td><td></td><td></td><td>70-90% ethyl alcohol</td></tr><tr><td>Tap water</td><td></td><td></td><td>Heated to 46-48 &amp;deg;C before use</td></tr><tr><td>Balance</td><td></td><td></td><td>Accurate to at least 0.1 g</td></tr><tr><td>Metal tray</td><td></td><td></td><td>Capacity 10 to 15 L, to collect the remaining digestive juice</td></tr><tr><td>Pipettes and pipette holder</td><td></td><td></td><td>Different sizes (1, 10 and 25 mL)</td></tr><tr><td>Thermometer</td><td></td><td></td><td>Accurate to 0.5 &amp;deg;C, temperature range at least from 20 &amp;deg; C to 70&amp;nbsp;&amp;deg;C</td></tr><tr><td>Siphon</td><td></td><td></td><td>For tap water</td></tr></tbody></table>

magnetic stirrer method for the detection of trichinella larvae in muscle samples

旋毛虫病是人类一种使人衰弱的疾病，被感染了旋毛虫属的线虫幼虫动物的生的或未煮熟的肉类消费量造成的。全球人类感染的最重要来源是野味及猪肉或猪肉制品。在许多国家，人类旋毛虫病的预防是基于肌肉样本的易感动物尸体的人工消化的方式识别受感染的动物。 有基于肉的消化，但磁力搅拌器方法被认为是金标准的几种方法。此方法允许肌肉样品和随后的过滤和沉降步骤的酶消化后的检测旋毛虫通过显微镜。虽然这种方法不需要特殊的和昂贵的设备，就不能使用的内部控制。因此，严格的质量管理应applie整个试验D。目前的工作的目的是要提供详细的操作说明和方法来分析的基础上，欧盟参考实验室寄生虫和旋毛虫德国国家参考实验室的经验的关键控制点。

<html>消化后，将感染性肌幼虫的形状可以变化，使得识别更加困难。典型形式包括紧密缠绕的幼虫，轻轻地盘绕幼虫或C形或完全解开幼虫（ 图2B - 2C）。每克发现幼虫的数目可以显着变化，并且可以从一个幼虫到几百幼虫范围。 感染性肌幼虫的形态示于图2A。 旋毛虫是0.7至1.5毫米长，宽度大约0.3mm。食管狭窄，有一个略显圆润结束。角质层是光滑的。在体腔内，该stichosome的前半部分，一个结构构成的细长管45至55的大细胞（stichocytes）一排包围的，可以观察到。直肠也是圆的，没有任何突出物或附属物10,11。 t"的&gt;其他的结构，除了旋毛虫 ，可以肌肉消化后发现的一些例子示于图3A。 - 3F野生动物常常感染其他寄生虫或可用于测试的肌肉样品中由动画的剜过程被污染或无生命的结构/生物。幼虫的长度，前部和后部端部的外观和的stichosome帮助的发生，以不同的线虫属12区分。 <img alt="图1" src="/files/ftp_upload/55354/55354fig1.jpg" /> 图1: 旋毛虫后镜检（A）不足和（B）有足够的漂洗步骤。刻度条表示100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55354/55354fig1large.jpg" target="_blank">请点击此处查看大图已经</a>这个数字rsion。 <img alt="图2" src="/files/ftp_upload/55354/55354fig2.jpg" /> 图2: 感染形态旋毛虫显示幼虫（A）的直肠，Stichosome和食道 。消化显示（B）后肌幼虫可能的形状紧密缠绕，轻轻地盘绕动的幼虫和（C）解开幼虫。刻度条表示100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55354/55354fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55354/55354fig3.jpg" /> 图3: 肌肉的消化样品中的磁力搅拌器方法后偶然发现的例子。 （A）的鬃毛般的头发野猪发现蚯蚓; （B）从野猪Alaria翅 ; （C）从野猪的肌纤维; （D）Metastrongylus藻。从野猪; 弓首 SP:（E）植物纤维野猪（F）中。幼虫从野猪。刻度条表示100μm左右。 <a href="http://ecsource.jove.com/files/ftp_upload/55354/55354fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples at JoVE.com

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

旋毛虫病的人在许多国家预防全球是基于消化，其中的磁力搅拌器方法被认为是黄金标准的方法，从易感动物肌肉样品的实验室检查。

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

magnetic-stirrer-method-for-detection-trichinella-larvae-muscle

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

28.6K 次观看.  Federal Institute for Risk Assessment. 旋毛虫病的人在许多国家预防全球是基于消化，其中的磁力搅拌器方法被认为是黄金标准的方法，从易感动物肌肉样品的实验室检查。 

视频: Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

鞭虫感染muris：在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

科研

免疫与感染

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

生物学

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

发育生物学

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

神经科学

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

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

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

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

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 ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

医学

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

对于检测磁力搅拌器方法

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此视频中的章节

鞭虫感染muris：在肠道内的2型免疫和炎症模型

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

检查斑马鱼肌肉疾病模型中的肌肉再生

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

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在收缩力测量评估肌肉功能在斑马鱼幼虫

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

对于检测磁力搅拌器方法

摘要

探索更多视频

此视频中的章节

鞭虫感染muris：在肠道内的2型免疫和炎症模型

ELIME霉菌毒素的检测方法（酶联免疫磁电化学）

检查斑马鱼肌肉疾病模型中的肌肉再生

肌肉注射沿运动终板:微创方法来接送示踪剂直接为运动神经元

去除

在收缩力测量评估肌肉功能在斑马鱼幼虫

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络