Madison Grant, Steven Foltz, Halie Zastre and Junna Luan provided technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number AR065077. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

所有的动物程序进行格鲁吉亚机构动物护理和使用委员会的大学动物下使用协议A2013 07-016（Beedle）的批准。 1.未固定骨骼肌冷冻保存<ol><li>制备<ol><li>切软木成小方块（约1cm×1厘米）用刀片，用细尖标记物到2-甲基丁烷耐识别源鼠标和肌肉，并进行了很浅的切口在软木写（约1毫米）在整个上表面上。将塑料盖玻片到剪切用于定向组织。重复，直到软木塞准备好要冷冻保存每一个组织。 </li><li>得到的液氮，2-甲基丁烷，包埋树脂，低温温度计，软木塞，清扫工具，以及研究小鼠。 注意:液氮遇热可能爆炸压缩气体。处理液体妮时，戴上白大褂，低温手套，面部防护特罗根;与皮肤接触或眼睛可能导致烧伤或冻伤。 2-甲基丁烷是一种易燃，有毒，健康和环境的危害。穿戴个人防护设备（实验室外套，手套，安全眼镜），在通风橱打开库存容器，所需的冷冻（一般在200至400毫升）少量转移至单独的容器中，可以紧密地封闭，并避免吸入。 </li><li>安乐死小鼠安乐死的麻醉下经批准的方法。简单地说，将鼠标与氧气2.5％，异氟醚从异氟醚蒸发吸入室，等到鼠标后20秒停止移动，检查踏板反射。当踏板反射是否定的，通过颈脱位6安乐死研究小鼠。 注:安乐死方法需要通过伦理委员会的批准。 </li><li>去除皮肤上覆远端后肢切只是细点狄氏脚踝上方末节前段筋ction剪刀。抓住用细镊子远端肌腱，轻轻拉起，朝膝盖，同时削减外侧筋膜释放肌肉。从膝盖垂线拉肌肉，同时使一裁终局摘除TA肌肉7。 注:TA肌肉在此用作一个例子，但任何小鼠骨骼肌或心脏可以在该协议如果适合于用户的实验目标被取代的的TA。唯一的限制是，组织必须足够小，以实现在其整个深度快速冷冻;建议1厘米×1厘米的最大组织的大小。 </li><li>重复在对面的腿，并剖析了要收集任何其他组织。尽可能快地进行清扫冷冻保存之前，以限制组织退化。 </li><li>东方每一块肌肉上的预标记的软木横截面。站肌肉垂直于软木塞与远侧肌腱触摸软木和肌肉分机的顶结束了，由盖玻片举行直立。 </li><li>尽快冷冻保存用于组织学分析的所有组织解剖后，优选在5分钟但绝对由于源动物安乐死经过不超过15分钟的时间。 </li></ol></li><li>冷冻保存<ol><li>开始解剖结束前冷却冷冻浴培养5 min。倾2-甲基丁烷到打开的金属烧杯中的大约3厘米深。倾液氮成隔热容器为2至4厘米的深度。设置2-甲基丁烷进入液氮的烧杯氮气就会开始沸腾。避免氮气溅入2-甲基丁烷烧杯中。 注意:准备在通风橱或通风良好的地方冻结浴。 </li><li>插入低温温度计插入2-甲基丁烷，监测温度并用叉子频繁搅拌以确保均匀的冷却。继续搅拌和冷却，直至2-MEthylbutane达到-140℃，加入新的液氮作为必要的外浴。 注:-140°C是最适宜的温度，以尽量减少横纹肌冰晶的文物;其它组织可在不同温度下最佳的冷冻保存（ 例如，脑，-90℃）。 </li><li>它满足了软木塞，并立即在-140℃的下降瓶塞入2-甲基肌肉的50％ - 嵌入树脂只低35应用。快速重复每批次冻结长达8个组织。搅拌30秒，刮烧杯的底部，以确保组织不冻结成固化2-甲基丁烷。 </li><li>用叉子，勺子开槽或大镊子从2-甲基拉各组织软木塞。快速去除盖玻片，轻拍掉多余的2甲基丁烷放入烧杯中，然后删除该组织软木塞到外氮浴。重复在烧杯中所有剩余的软木塞。 </li><li>在液氮或干冰传输样本-80℃的冷冻储存。 </li><li>如果有任何组织仍是冷冻保存，重复步骤1.2.3至1.2.4。添加其他2-甲基烧杯或液氮外浴必要再冷却至-140℃。处理用过的2甲基丁烷作为危险废物处理的。 </li></ol></li></ol> 2.收集冰冻切片组织学和RNA应用<ol><li>制备。 <ol><li>在分析天平上预称重一个DNA酶/无RNase蒸压管。然后，进入低温恒温室预冷。重复，直到管是准备要收集所有池冷冻切片样本。 </li><li>对于骨骼肌切片，确认低温恒温室温度是其内部温度-21至-22℃。传输任何容器用面巾纸将切入腔和允许容器（S）平衡的低温恒温器温度开幕前至少15分钟。 </li><li>插入一个新的一次性刀片的低温恒温器。另外，除去现有的刀片，用RNase去污液喷洒，用的DDH 2 O冲洗，并重新插入低温恒温器冷却。喷干净的纸巾用70％乙醇和仔细擦拭刀片和刀片夹紧平台。 <ol><li>喷干净的纸巾用核糖核酸酶净化解决方案，并擦拭低温恒温器刷。喷雾组织与DDH 2 O的，再次擦拭画笔，设置它们在低温恒温室中在清洁的表面。 小心:低温恒温器刀片应由护刀在不使用时覆盖。此外，核糖核酸酶去污溶液是有毒的，将冻结，以形成在冷低温恒温室中的沉淀物。因此，谨慎操作，避免其在低温恒温室中使用。 </li></ol></li><li>在低温恒温器室，将一个硬币大小的一滴树脂嵌入到一个温暖的标本盘（约300微升），对树脂的顶部，请按设置组织软木下来，然后在FR设定夹头eezing铁路或到快速冻结的元素（如果可用）。 </li><li>嵌入树脂固化（白色）后，对软木的顶部添加额外的嵌入树脂，周围组织的低35％，按热提取到嵌入树脂用于快速冻结，以更好地稳定该组织。切片以使树脂充分硬化之前等待5分钟。 </li><li>保证在试样夹处于其最缩回位置，从叶片最远。插入组织标本夹头入试样夹具。 </li></ol></li><li>冷冻切片准备。 <ol><li>松开刀架保持件，刀片的间隙角设为10°（或适合所使用的刀片载体的角度），并重新拧紧。松开刹车，转动手轮，以降低对刀片的肌肉。估计组织和叶片之间的最近距离，然后将组织脱离刀片和接合手轮刹车。 </li><li>松开高度调节LEVEr和向前或向后移动至刀片载体，分别，如果组织的端部是从叶片超过约2mm，或如果刀片撞击组织。拧紧并再次降低组织从刀片检查距离。重复调整，直到叶片是从由视觉估计的组织的端部1到2毫米。 </li><li>降低对刀片的组织和评估组织角度横截面。锁定手轮并释放标本夹杆。推样品夹钳左或右，直到组织的水平方向是垂直于刀片。 </li><li>推试样夹持向上或向下调节"y"的方向，使组织切片将垂直于肌肉的长轴。拧紧标本夹杆。 </li><li>使用过程和精细的前馈以超前的标本，直到它刚好接触到刀片。如果从一个特定的组织深度求的部分，部分厚度之和复位到零（组织的顶部）。 </li><li>在30微米设置低温恒温器与部分厚度微调功能。切割和直到达到组织收集预选深度丢弃的部分（ 例如，400微米从顶部）。 </li></ol></li><li>收集提取RNA的冰冻切片。 <ol><li>打开管收集区，并将其放置刀架附近。使用预冷却，清洁刷，因为它是从刀片切成每节拾取和冷冻切片转移到管中。重复，直到汇集部分称重30毫克或达到期望的组织深度。 注:对于成年小鼠胫前，约400至4000微米的组织深度集合通常产生25 - 40毫克。使用金属钳区段转移到收集管不推荐为部分倾向于粘和结块在金属表面上。 </li><li>如果嵌入树脂包围肌肉冷冻切片，锁定手刹用剃刀刀片剃掉小块树脂的直到只有周围肌肉的顶部薄层。与刀片切割时总是树脂角度从肌肉了。 注:嵌入树脂基本上不损害下游RNA制备的薄层。如果较厚的嵌入树脂的存在，用刷子将冷冻切片移动到收集管前逗它从肌肉了。 </li><li>或者，在批量到集合管上的叶片的载体和转移的池部。 注:然而，这种方法往往是比较慢，并且部分是更有可能坚持和聚集在一起，从而可以减少在后续步骤针同质化的效率。 </li><li>快速地将汇集的冷冻切片管成分析天平和记录管的重量。紧随管返回寒冷低温恒温室保持接近-20°C截面温度。计算汇集的部分的重量。 注:如果RNA分离将在广告出现ifferent天，在-80存储汇集冷冻切片管℃直至使用。 </li></ol></li><li>收集的冰冻切片组织学检查。 <ol><li>按部分厚度按钮细切片和用箭头在低温恒温器切片厚度设置为7微米（或其它适当部分的厚度，一般为6至10微米）。 注:稀释剂切片（6至10微米）应当用于组织学应用，以确保染色试剂可以穿透组织部分的深度。薄的部分可以从任何深度cryosectioning期间进行，但更深部分是优选的，因为包埋树脂，其与组织深度的增加，不会损害组织染色。 </li><li>切割并丢弃4至7段，以获得一致的，即使组织表面。记下组织的深度。 </li><li>切部分，并将其定向刀片载体的表面上。通过快速并轻轻触摸温暖（室温）显微镜载玻片拿起部到刀片载体上的部分。返回滑动到室温。继续，直到获得所需的幻灯片的数量。记下最终组织的深度。 注:收集每个组织第二次（重复）部分建议。 </li><li>随着切片完整，搞手轮制动，标本回到后方最大位置，并取下组织夹头。使用低温恒温器热元件以熔化保持组织软木对试样夹头嵌入树脂。删除组织软木塞，用纸巾擦拭，并返回到存储。 </li><li>从步骤2.1.2其余每个组织重复。允许的最后一个组织被安装后载玻片干燥20分钟。然后，直到需要在滑动箱用于组织学或免疫荧光染色或冷冻载玻片在-80℃。 </li></ol></li></ol> 3.从冷冻切片汇集RNA分离<ol><li> RNA提取<ol><li>汇集冷冻切片以冰的移动管（多个）。立即添加有机RNA提取试剂以每50毫克的重量冰冻切片约1ml试剂，每管600通常微升的比率。 注意:RNA提取试剂是有毒的，腐蚀性和刺激性。按照制造商的安全操作规程。 </li><li>用1ml注射器用18号针头，画出RNA提取液和直到所有组织悬浮于该溶液冲洗该管的壁。试针均化期间，以尽量减少气泡。 </li><li>用针尖捣碎和分散任何成群的冰冻切片和碎片粘附在管壁上。磨碎由上下使样品通过针对五笔均匀化，然后将样品返回到冰上。重复这两个步骤的三到五倍，或多次需要中断所有团块并通过针容易地通过样品。 </li><li>取出18号针，并用22号针进行更换。仔细TRiturate样品为五笔，然后将样品返回到冰上。重复研磨的三到四倍，以达到一个非常细分散的组织匀浆。 注意:如果组织颗粒开始绘制了样品时阻止的针，从注射器排出的所有样品并切换回18号针头进行进一步均质化。可以加入用25或26号针头的额外研磨步骤，以获得最大的产量。然而，针可以被堵塞所以有样品溢出的危险。 </li><li>移动冰冷到室温的样品。孵育5分钟以破坏分子的复合物。 </li><li>在通风橱中，加入0.1毫升，每1毫升RNA分离试剂使用1-溴-3-氯戊烷（BCP）（步骤3.1.1）的，通常为60微升每管。大力摇晃管手，持续15秒（不涡）。在室温下孵育2至3分钟的样品。然后，离心以12,000 xg离心15分钟，4℃。 警告: </st荣&gt; BCP是易燃性和毒性。处理在通风橱和戴手套，实验室外套，和护目镜。 </li><li>仔细收集上层水相（无色）含有RNA，并将其转移到一个干净的试管。添加70％乙醇（于DEPC水中）和涡流混合等体积。在室温下孵育达10分钟。 注意:中间间期和低酚-BCP相可为分别的基因组DNA和蛋白质，按照生产商的指示或在适当的处置酚-BCP有害废物容器收集处理。 </li></ol></li><li> RNA纯化。 <ol><li>遵循制造商的样品结合，洗涤和洗脱用8小的变化的指示。例如: </li><li>放置DNA酶/无RNase水的等分试样到37℃浴或培养箱预先温暖步骤3.2.4。 </li><li>从3.1.8添加RNA样品上方纯化柱并离心样品15秒在室温下以12000 xg离心。倾通过放回同一列并重新离心的流动。重复一个附加的时间来最大化RNA结合，然后丢弃通过最终流。 </li><li>根据制造商的说明8执行洗涤步骤。适用的洗涤缓冲液700微升（无乙醇）到塔，旋转15秒以12,000 xg离心，并弃去通过流。 </li><li>添加500μl的洗涤缓冲液（用乙醇）的向列中，自旋15秒以12,000 xg离心，并弃去通过流。重复此步骤一次。 </li><li>旋空柱1分钟，以12,000 xg离心以干燥膜。 </li><li>离心柱转移到一个干净无RNA酶，DNA酶管。添加的预热DNA酶/无RNA酶的水40微升（从3.2.1）到列膜的中央。在室温下，孵育2分钟，然后以12,000×g下离心2分钟。 注:40微升洗脱体积是原来的每毫克约1.3微升组织30毫克汇集冰冻切片的。调整为适合不同的起始组织重量的洗脱体积，但不降低低于32微升。第二个洗脱，使用每原始组织毫克约0.7微升，可以加入，以增加总RNA产量。 </li><li>用分光光度计，电泳和/或生物分析仪分析浓度和纯度的RNA（柱洗脱）。直到所需的下游应用，如用于定量PCR 4逆转录在-80℃下储存的RNA。 注:在下游应用使用RNA前，建议使用DNase处理。可以下列柱洗脱洗涤步骤之前在某些RNA纯化柱进行这种处理，在此特定点，或在RNA中任下游应用的第一步的等分试样。所述RNA应几年是稳定的。 </li></ol></li></ol> 4.组织学分析免疫荧光Stainin摹肌肉的冰冻切片<ol><li>简单的免疫协议。 <ol><li>使用疏水用笔圈出组部分的每张幻灯片。轻轻放下PBS成圆圈区域（约80微升对于小面积达500微升了很大的表面积），注意不要触碰组织。孵育在室温下5分钟。 注意:如果使用冷冻幻灯片，除去从-80℃冷冻载玻片并在室温下孵育20至30分钟，以解冻和干燥，然后按上述进行。至少需要两个幻灯片:一是实验幻灯片在小学和中学抗体孵育;并为其中一抗一个控制幻灯片被排除在外，"二次只有"控制。 </li><li>提示幻灯片以倒出PBS。添加5％驴血清的PBS（封闭溶液）到盘旋区;要小心，以保证肌肉部分没有干出来的。在室温下孵育30分钟（或最多几个小时在湿度室中）。 </li><li>制备初级抗体溶液来分析肌肉再生。混合封闭溶液，用eMHC抗体（1:40）和ColVI抗体（1:1000）。准备约150微升，300微升或500微升为小型，中型或大型滑坡区，分别为。涡旋5秒，然后以15,000 xg离心离心2分钟以沉淀任何沉淀物。 </li><li>对于实验幻灯片，小费幻灯片倒掉封闭液，然后从4.1.3添加的主要抗体溶液。对于辅助控制滑，小费幻灯片倒掉封闭液，然后加入新鲜的封闭液（无抗体）。在4℃孵育在湿度室载玻片过夜。 注意:当吹打抗体溶液在载玻片上，始终从初级抗体溶液管的顶部拉液体，不破坏在管子的底部的任何沉淀物。 </li><li>提示滑动以倒出的解决方案。 PBS滴添加到每张幻灯片的圆圈区域进行冲洗，尖倒关闭，然后添加更多的PBS中并孵育3至5分钟。重复总共洗涤三次。 注:洗涤时间是相当灵活的，如果需要的话可以加长到20分钟。通常，溶液变化的数量比时间更重要。 </li><li> 500稀释红色和绿色荧光偶联的第二抗体来检测小鼠IgG1（eMHC）和兔IgG（ColVI）和1:10000稀释的DAPI核用1准备所有幻灯片二级抗体溶液（包括次级控制幻灯片）弄脏。 <ol><li>例如，将1微升的DAPI与9微升的DDH 2 O，使1:10稀释的DAPI。然后，对于500微升终体积中，添加1.0微升的抗小鼠IgG1红色+ 1.0微升抗兔IgG-绿色+ 0.5微升1:10的DAPI至447.5微升阻断溶液。涡旋以15,000 xg离心混合和离心2分钟以沉淀任何沉淀物。 注:理想地，所有第二抗体应当从同一宿主物种。 </li></ol></li><li>提示幻灯片倒了最后PBS洗。添加二抗溶液覆盖所有的组织。覆盖玻片，以保护他们免受光，并在室温温育30分钟。 注:所有的二抗应当验证，以有双标记实验等品种最小的交叉反应。 </li><li>在4.1.5中描述洗的幻灯片。最后一次洗涤后，小费幻灯片倒出PBS，并设置组织的幻灯片。添加水性安装介质的3至4滴沿一侧。 </li><li>玻璃盖玻片的边缘设置只是到安装媒体的外边缘。使用镊子，慢慢放下盖玻片向薄纸，然后，松开盖玻片，轻轻地拍打到位。最后，轻轻按下盖玻片的每个角落以使其稳定。 </li><li>避光和存储的玻片在4℃直至使用。 注:对于长期储存，沿着次边申请指甲油薄薄的一层Ë盖玻片，以帮助防止干燥的幻灯片。 </li></ol></li><li>组织学评价。 <ol><li>图片使用啶或共聚焦显微镜用适当的过滤器的幻灯片检测eMHC（红色）; ColVI（绿色）;和DAPI染色的细胞核（蓝色），通常使用20X物镜1,5。 </li><li>确认从实验幻灯片的荧光信号是从辅助控制幻灯片不同来演示eMHC和ColVI检测4的特异性。 注:肌肉损伤后和可变地与肌营养不良小鼠eMHC阳性再生纤维应该是可见的，从约2至7天。胶原VI是存在于周围的肌肉纤维，血管和神经的细胞外基质和在围和肌外膜较大结缔组织束。 DAPI核染色是有用的识别与中央核与核周围的肌肉纤维，指示纤维再生，而我的存在nfiltrating细胞。坏死或受伤的纤维可与鼠IgG二级抗体弱染色由于检测内源性的IgG通过受损肌肉细胞膜穿透纤维。 </li><li>为了量化再生，采取重叠的显微镜照片与整个图像中的每个荧光滤光器。合并eMHC，ColVI和DAPI图像的每个位置，然后对齐图片重建地图整款1,5。 </li><li>使用分析软件，计数eMHC阳性纤维的数量和总的纤维来计算最近再生（100 *（＃eMHC +纤维/＃总纤维））的数量。此外，计数集中核纤维数出的总纤维的测量再生在较长时期内（100 *（＃CN +纤维/＃总纤维））1,5。 </li></ol></li></ol>

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The author declares that she has no competing financial interests.

为了实现用这种方法最好的结果，保持组织冷冻期间包埋树脂限制在低三分之一或一半的肌肉因为过量的树脂会减缓汇集冷冻切片的集合，并且可以增加在RNA分离包埋树脂污染。另外，针均化期间仔细注意重要的是最大限度地提高产量和最小化堵塞针的概率。该协议可以通过使用一个Luer-Lok接头的注射器，以防止样品的损失，如果在针被阻塞并且需要高压以移去堵塞进行修改。还可以加入具有25或...

该技术的目的是通过不同的方式，如组织学和基因表达，从一个单一的小骨骼肌源组织访问进行多次实验的分析。显微镜应用是最敏感的样品的保存方法，其必须小心地控制冷冻保存期间限制冰晶工件的形成。因此，方法的发展是基于胫骨前肌（TA）肌肉冻部分覆盖在-140℃的液氮冷却的2-甲基丁烷浴为免疫荧光显微镜和基因表达分析源材料嵌入树脂。 用于多样的技术方法相同的源材料，需要的是用于肌内注射的基于实验，其中左和右的肌肉代表不同条件，一是实验和一个控制特别重要。例如，在肌肉再生研究，一亩SCLE与毒素注射引起广泛组织损害而对侧肌肉用作媒介物注射的对照1。同样，对于肌肉疾病的基因治疗研究通常用肌肉注射的基因治疗载体的有效性开始与空载体，无关向量或车辆控制在对侧2进行比较。因此，这是不可能的每个的TA肌肉源到不同的应用程序。 公共策略来处理这个问题是:i）使用不同的肌肉群的每个应用程序，ⅱ）使用额外的小鼠，或iii）切断一块肌肉为每个应用程序的。然而，肌肉群之间相当大的差异，很难将数据从单独的应用程序进行比较，并附加动物增加费用，并且如果其他方法存在不良有道理的。除肌肉解剖后到源不同的应用程序是最好的选择我N多的情况。然而，肌片往往太小使用粉碎液氮或机械研磨技术下均质化2-5。肌肉是填充有细胞外基质和收缩蛋白高度的结构组织，机械均化不足导致的随后的DNA，RNA或蛋白质的低的产率。这里详述该方法允许少量的组织从一个源的肌肉在多个应用程序使用，并cryosectioning和针研磨列入改善了更好的RNA产量机械同质化。

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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Cork</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">23420-708</td>
			<td style="width:259px;">Cut into small squares with a sharp blade.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Plastic coverslip</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:115px;">12-547</td>
			<td style="width:259px;">Used to orient the muscle during freezing.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Low temperature thermometer</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">89370-158</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">2-methylbutane</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">M32631-4L</td>
			<td style="width:259px;">Caution: hazardous chemical. Store in flammable cabinet.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Embedding resin: &#34;cryomatrix&#34;</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">6769006</td>
			<td style="width:259px;">Other embedding resins can be substituted for cryomatrix.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Cryostat</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">microm HM550 with disposable blade carrier</td>
			<td style="width:259px;">Any working cryostat should be sufficient for the protocol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Disposable cryostat blade</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">3052835</td>
			<td style="width:259px;">Use an appropriate blade or knife for the cryostat to be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">RNAse decontamination solution: &#34;RNase Zap&#34;</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM9780</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Analytical balance</td>
			<td style="width:128px;">Mettler Toledo</td>
			<td style="width:115px;">XS64</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Paint brush</td>
			<td style="width:128px;">Daler Rowney</td>
			<td style="width:115px;">214900920</td>
			<td style="width:259px;">Use to handle cryosections. Can be found with in stores with simple art supplies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Razor blade</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">55411-050</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Microscope slide</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">48311600</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">RNA organic extraction reagent: TRIzol</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">15596026</td>
			<td style="width:259px;">Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">18 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305185</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">22 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305155</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">26 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305115</td>
			<td style="width:259px;">Optional. Can be used for a third round of sample trituration in the RNA extraction protocol.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">1 mL syringe</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD309659</td>
			<td style="width:259px;">For very high value samples, a luer-lok syringe is recommended (e.g. VWR BD309628).</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">1-bromo-3-chloropentane (BCP)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">B9673</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">For 70% ethanol in DEPC water: 200 proof alcohol</td>
			<td style="width:128px;">Decon Laboratories, Inc.</td>
			<td style="width:115px;">+M18027161M</td>
			<td style="width:259px;">Mix 35 ml 200 proof alcohol + 15 mL DEPC water.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">For 70% ethanol in DEPC water: DEPC-treated water</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM9922</td>
			<td style="width:259px;">Mix 35 ml 200 proof alcohol + 15 mL DEPC water.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">RNA purification kit: PureLink RNA minikit</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">12183018A</td>
			<td style="width:259px;">Final steps of RNA preparation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">DNase/Rnase-free water&#160;</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:115px;">10977</td>
			<td style="width:259px;">DEPC-treated water can also be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Spectrophotometer: Nanodrop 2000</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">NanoDrop 2000</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Dnase I</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM2222</td>
			<td style="width:259px;">Treat purified RNA to remove any DNA contamination before downstream appications.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Hydrophobic pen</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">8899</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dulbecco&#39;s PBS</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:115px;">14190</td>
			<td style="width:259px;">PBS for immunofluorescence protocol.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">Donkey serum</td>
			<td style="width:128px;">Jackson ImmunoResearch Laoratories, Inc</td>
			<td style="width:115px;">017-000-121</td>
			<td style="width:259px;">Rehydrate normal donkey serum stock according to the manufacturer&#39;s instructions, then dilute an aliquot to 5% for immunofluorescence.&#160; Normal goat serum can also be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">eMHC antibody</td>
			<td style="width:128px;">University of Iowa Developmental Studies Hybridoma Bank</td>
			<td style="width:115px;">F1.652</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Collagen VI antibody</td>
			<td style="width:128px;">Fitzgerald Industries</td>
			<td style="width:115px;">#70R-CR009x</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Donkey anti-rabbit AlexaFluor488</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">A21206</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Goat anti-mouse IgG1 AlexaFluor546</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td>A21123</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">D1306</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Aqueous mounting media: Permafluor</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">TA-030-FM</td>
			<td style="width:259px;">Only use mounting media designed for fluorescent applications with anti-fade properties.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Glass coverslip</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">16004-314</td>
			<td style="width:259px;">Use for mounting slides at the end of immunofluorescence protocl</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">Image analysis software: ImageProExpress</td>
			<td style="width:128px;">Media Cybernetics, Inc.</td>
			<td style="width:115px;">Image-Pro Express, or more advanced products</td>
			<td style="width:259px;">Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process.&#160; E.g. ImageProPremier.&#160;&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Merge and map section images: Photoshop</td>
			<td style="width:128px;">Adobe</td>
			<td style="width:115px;">Photoshop</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:135px;">Cardiotoxin</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">C9659</td>
			<td style="width:259px;">Sigma C9659 has been discontinued. Other options for cardiotoxin are EMD Millipore #217503; American Custom Chemicals Corp. # BIO0000618; or Ge Script # RP17303; but these have not been validated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">reverse transcription kit: Superscript III First-strand synthesis system</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">18080051</td>
			<td style="width:259px;">Any validated, high quality reverse transcription reagents can be used.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Standard PCR: GoTaq Flexi polymerase system</td>
			<td style="width:128px;">Promega</td>
			<td style="width:115px;">M8298</td>
			<td style="width:259px;">Any validated, high quality Taq polymerase system can be used. If DNA sequencing is to be used for any application downstream of the PCR, then a high fidelity PCR system should be used instead.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">SYBR green</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">S7585</td>
			<td style="width:259px;">For use in qPCR when not using a dedicated qPCR master mix. Use with SuperROX (for Applied Biosystems instruments) and GoTaq Flexi polymerase and buffers.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">ROX: SuperROX, 15 mM</td>
			<td style="width:128px;">BioResearch Technologies, Inc. Novato CA</td>
			<td style="width:115px;">SR-1000-10</td>
			<td style="width:259px;">SuperROX is more stable in the PCR reaction, so it is preferred for use as a qPCR passive reference dye over ROX (carboxy-X-rhodamine). For qPCR with Applied Biosystems instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Real-time PCR</td>
			<td style="width:128px;">Applied Biosystems</td>
			<td style="width:115px;">7900HT</td>
			<td style="width:259px;"></td>
		</tr>
	</tbody>
</table>

cryosectioning of contiguous regions of a single mouse skeletal muscle for gene expression and histological analyses

用这种方法，连续冷冻切片被收集，以使对组织的组织学和RNA的富集基因的表达使用相邻区域从单个小鼠骨骼肌既显微镜应用。通常情况下，它是具有挑战性的，实现小骨骼肌样品充分均质化，因为缓冲器的卷可能太低高效研磨应用中，但没有足够的机械破碎，缓冲试剂的肌肉限制渗透的致密组织架构，最终导致低的RNA产量。按照此处所报告的协议，30微米的部分被收集并汇集允许cryosectioning和随后的针均化以机械地扰乱肌肉，增加暴露缓冲渗透的表面积。该技术的主要限制是，它需要一个低温恒温器，它是相对较低的吞吐量。然而，高质量的RNA可从汇集M个小样品中获得uscle冷冻切片，使得这种方法对于许多不同的骨骼肌肉和其他组织访问。此外，这种技术使得能够匹配分析（ 例如，组织病理学和基因表达），以便测量可以跨应用程序，以减少实验的不确定性，并减少所需的复制动物实验，以源为一个小的组织直接比较由单一骨骼肌的相邻区域多个应用程序。

肌肉冷冻切片RNA质量高，并提供足够的产量为大多数应用十六骨骼肌RNA制剂分析显示在使用池胫骨前（TA）肌的19.4〜41毫克，从8对照组小鼠表1。两个左（L）和在再生实验中制备与收集的25μl的生理盐水或10μM的心脏毒素的纵向肌内注射后第3天以引起使用方法肌肉损伤肌肉右（R）的TA肌肉先前报道1。如?...

Watch this Scientific Journal Video about Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses at JoVE.com

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

连续的低温部分被收集，以便利用邻近地区从一个单一的小鼠骨骼肌基因表达测量组织的应用程序和RNA的富集。 30毫克汇集冷冻切片和测量的应用程序之间直接比较 - 高品质的RNA是从20获得。

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

cryosectioning-contiguous-regions-single-mouse-skeletal-muscle-for

Research

JoVE Journal

Biology

15.6K 次观看.  University of Georgia. 连续的低温部分被收集，以便利用邻近地区从一个单一的小鼠骨骼肌基因表达测量组织的应用程序和RNA的富集。 30毫克汇集冷冻切片和测量的应用程序之间直接比较 - 高品质的RNA是从20获得。 

视频: Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.