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>

<ol>
	<li>Foltz, S. J., 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=Abnormal+skeletal+muscle+regeneration+plus+mild+alterations+in+mature+fiber+type+specification+in+Fktn-Deficient+Dystroglycanopathy+Muscular+Dystrophy+Mice.">Abnormal skeletal muscle regeneration plus mild alterations in mature fiber type specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.</a> PLoS One. 11 (1), 0147049 (2016).</li><li>Bartoli, M., 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=Noninvasive+monitoring+of+therapeutic+gene+transfer+in+animal+models+of+muscular+dystrophies.">Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies.</a> Gene Ther. 13, 20-28 (2006).</li><li>Ip, W. T., Huggins, C. E., Pepe, S., Delbridge, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+RNA+preparation+options+for+archived+myocardial+biopsies.">Evaluating RNA preparation options for archived myocardial biopsies.</a> Heart Lung Circ. 20 (5), 329-331 (2011).</li><li>Beedle, A. M., 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=Mouse+fukutin+deletion+impairs+dystroglycan+processing+and+recapitulates+muscular+dystrophy.">Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy.</a> J. Clin. Invest. 122 (9), 3330-3342 (2012).</li><li>Foltz, S. J., 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=Four-week+rapamycin+treatment+improves+muscular+dystrophy+in+a+fukutin-deficient+mouse+model+of+dystroglycanopathy.">Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy.</a> Skeletal Muscle. 6, 20 (2016).</li><li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting.">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nat. Protoc. 10, 1612-1624 (2015).</li><li>Rump, L. V., Asamoah, B., Gonzalez-Escalona, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+commercial+RNA+extraction+kits+for+preparation+of+DNA-free+total+RNA+from+Salmonella+cells.">Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.</a> BMC Res. Notes. 3, 211 (2010).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> J. Vis. Exp. (45), e2565 (2010).</li><li>Valdez, M. R., Richardson, J. A., Klein, W. H., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+Myf5+to+support+myogenic+differentiation+without+myogenin,+MyoD,+and+MRF4.">Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4.</a> Dev Biol. 219 (2), 287-298 (2000).</li><li>Pavlath, G. K., Dominov, J. A., Kegley, K. M., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+transgenic+skeletal+muscles+with+altered+timing+of+expression+of+the+basic+helix-loop-helix+muscle+regulatory+factor+MRF4.">Regeneration of transgenic skeletal muscles with altered timing of expression of the basic helix-loop-helix muscle regulatory factor MRF4.</a> Am. J. Pathol. 162 (5), 1685-1691 (2003).</li><li>Chomczynski, P., Mackey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+chloroform+by+bromo-chloropropane+in+the+single-step+method+of+RNA+isolation.">Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation.</a> Anal. Biochem. 225 (1), 163-164 (1995).</li><li>Lee, J. T. Y., Cheung, K. M. C., Leung, V. Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+RNA+from+tough+tissues+with+high+proteoglycan+content+by+cryosection,+second+phase+separation+and+high+salt+precipitation.">Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation.</a> J. Biol. Methods. 2 (2), 20 (2015).</li><li>Guo, D., Catchpoole, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+RNA+following+cryosections+of+archived+frozen+tissue.">Isolation of intact RNA following cryosections of archived frozen tissue.</a> BioTechniques. 34, 48-50 (2003).</li><li>Goodpaster, T., Randolph-Habecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flexible+mouse-on-mouse+immunohistochemical+staining+technique+adaptable+to+biotin-free+reagents,+immunofluorescence,+and+multiple+antibody+staining.">A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining.</a> J. Histochem. Cytochem. 62 (3), 197-204 (2014).</li><li>Ikezawa, M., Minami, N., Takahashi, M., Goto, Y., Miike, T., Nonaka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+gene+analysis+of+130+patients+with+Duchenne+muscular+dystrophy+with+a+special+reference+to+muscle+mRNA+analysis.">Dystrophin gene analysis of 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA analysis.</a> Brain Dev. 20 (3), 165-168 (1998).</li><li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle.">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Anal. Biochem. 474, 25-27 (2015).</li><li>Espina, V., Heiby, M., Pieroban, M., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+technology.">Laser capture microdissection technology.</a> Expert Rev Mol Diagnostics. 7 (5), 647-657 (2007).</li></ol>

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26号针的额外针均化步骤，以产生更精细的组织悬浮液来进一步提高RNA的产率。而氯仿可以代替BCP，这是不推荐的BCP是毒性较低和RNA 12的有机提取过程中导致较低水平的在水相中的基因组DNA污染。增加也没有建议合并冷冻切片用30微米的切片厚度为均质化效率会降低。 如果RNA的产率低于所需水平，各种策略可用于增加回收，例如:ⅰ）增加起始材料，以增加可能收率的毫克量; ⅱ）减少到低于30微米的切片厚度，以提高组织的机械均匀化; ⅲ）增加样品孵育和针均化的持续时间在该有机萃取剂，以提高机械和化学组织破坏;和iv）如组织块仍然存在，执行更严格的同质化针第二次提取步骤。交替，有可能是组织特异性的考虑，例如用于具有高的蛋白多糖含量13个样品的附加的相分离和沉淀步骤。在RNA的柱纯化，较大的洗脱体积可使用并进行第二次洗脱可以マximize总RNA回收，但在RNA浓度为代价。 A-柱后乙醇沉淀可用于浓缩所述RNA如果低浓度是与本变形例的关注。如果RNA降解是一个问题，解剖过程中减少的时间来冷冻保存，低温恒温器的表面和工具的更严格的清洗，以尽量减少RNA酶接触，执行在冷室中的针均质化步骤，除了RNase抑制剂试剂的冷冻切片14，以及频繁更换无RNase的解决方案，可以帮助各防止或减少暴露于RNA酶，减少切割活性。这是可能的简要沐浴所述组织中的RNA酶抑制剂的试剂解剖后，但冷冻保存之前，可以进一步降低样品降解。然而，应进行初步实验，以确保任何这种治疗并不增加冷冻保存过程中冰晶或其他工件。 虽然胚胎肌球蛋白重链/胶原VI间接免疫荧光在这里被用作对损伤的肌肉分析一个例子，薄的冷冻切片安装在显微镜从这些实验可用于任何相关的组织学染色，可以在冷冻切片中进行，包括具有交免疫荧光技术滑动-fixation和苏木/伊红染色。的确，改编到这里提供的简单免疫协议可能是必要的。例如，使用抗小鼠二级抗体来检测小鼠初级抗体（ 例如，eMHC）也可以检测在靶组织中的内源性小鼠免疫球蛋白。这样的内源性抗体一般积聚在受损或坏死的肌肉纤维损伤或肌营养不良造成的背景免疫荧光染色。一种二次控制滑动件（省略一次抗体）应始终进行检查，以评定染色的特异性。如果源抗体的背景是有问题的，块前步骤应该是添加到协议，以防止或最小化的内源性小鼠免疫球蛋白15的检测。 该方法的主要限制是，它需要一个低温恒温器，它是耗时的，这使得它相对低的吞吐量。例如，在该技术的专家能在约9至10小时以处理高达为汇集冷冻切片和显微镜载玻片16的肌肉（8玻片与所有16个组织的重复节）。新手cryosectioning，从2到4个样本池冰冻切片收集可合理低温恒温器的训练和一个或两个训练课程后掌握，相反，获得优质的冰冻切片组织学了更多的经验。因此，设备，时间，或训练因素可能使这种方法对于软组织，可以是用手动杵均化以及均匀化不太有用。 在与非低温恒温器均化的方法比较，横纹肌RNA制备小号已从肌肉活检报告为0.05 RNA产量肌肉16的0.7微克的RNA预毫克，每块肌肉17毫克最近0.27到1.08微克的RNA。因此，此处所描述的技术提供了RNA产量为比非低温恒温器的方法与使成对组织从同一样品的邻接区域分析的附加优点一样好或更好。值得注意的是，先前的研究也用在脊椎组织cryosectioning的同质化，同样发现cryosectioning组织提高效率的同质化RNA分离13。当这种技术牛骨骼肌样品中进行了测试，每个样品制备了平均的RNA产率是4.09±0.36微克，在这里13报告正常范围的低端。激光捕获显微切割是组织收集从冷冻切片RNA提取另一种选择。激光捕获优于在此汇集冷冻切片方法它允许特异性仅收集从部分细胞的期望子集，它可以在一个单一的组织切片达50微米厚的18来执行。然而，微解剖样品的收集可能是困难和合适的设备是不广泛可用，使得汇集冷冻切片均化到研究人员更容易获得。当这两种方法可供选择，偏好来分析组织的次区域为一个应用程序仅需要小分子RNA的数量将有利于激光捕获显微切割，同时汇集冷冻切片同质化是最好的时候，分区域分析是不太重要和需要RNA的更高的数量。 虽然组织学和RNA分离方法是这里的重点，汇集的冷冻切片的方法是很容易适应印迹分析或酶活性测量准备蛋白裂解物。例如，从心脏汇集冷冻切片溶解于蛋白质印迹分析4一第二从TA汇集冰冻切片匀浆线粒体功能5琥珀酸脱氢酶活性测定。可替代地，基因组DNA和蛋白质部分可以由其他相后RNA分离有机提取期间隔开，提供一个单一的低温恒温器会话之后导出从单一组织的基因组DNA，蛋白质，RNA和组织学测量的潜力。 总体而言，该方法的主要优点是通过使需要从单一组织不同样品制备多种分析方法来增加实验灵活性。该方法是最适合于肌肉和其它组织具有广泛的细胞内或细胞外结构降低基于杵组织均匀化的效率。

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。

<table><tbody><tr><td>Cork</td><td>VWR Scientific</td><td>23420-708</td><td>Cut into small squares with a sharp blade.</td></tr><tr><td>Plastic coverslip</td><td>Fisher Scientific</td><td>12-547</td><td>Used to orient the muscle during freezing.</td></tr><tr><td>Low temperature thermometer</td><td>VWR Scientific</td><td>89370-158</td><td></td></tr><tr><td>2-methylbutane</td><td>Sigma</td><td>M32631-4L</td><td>Caution: hazardous chemical. Store in flammable cabinet.</td></tr><tr><td>Embedding resin: &quot;cryomatrix&quot;</td><td>Thermo Fisher Scientific</td><td>6769006</td><td>Other embedding resins can be substituted for cryomatrix.</td></tr><tr><td>Cryostat</td><td>Thermo Fisher Scientific</td><td>microm HM550 with disposable blade carrier</td><td>Any working cryostat should be sufficient for the protocol.</td></tr><tr><td>Disposable cryostat blade</td><td>Thermo Fisher Scientific</td><td>3052835</td><td>Use an appropriate blade or knife for the cryostat to be used.</td></tr><tr><td>RNAse decontamination solution: &quot;RNase Zap&quot;</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>XS64</td><td></td></tr><tr><td>Paint brush</td><td>Daler Rowney</td><td>214900920</td><td>Use to handle cryosections. Can be found with in stores with simple art supplies.</td></tr><tr><td>Razor blade</td><td>VWR Scientific</td><td>55411-050</td><td></td></tr><tr><td>Microscope slide</td><td>VWR Scientific</td><td>48311600</td><td></td></tr><tr><td>RNA organic extraction reagent: TRIzol</td><td>Thermo Fisher Scientific</td><td>15596026</td><td>Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle.</td></tr><tr><td>18 gauge needle</td><td>VWR Scientific</td><td>BD305185</td><td></td></tr><tr><td>22 gauge needle</td><td>VWR Scientific</td><td>BD305155</td><td></td></tr><tr><td>26 gauge needle</td><td>VWR Scientific</td><td>BD305115</td><td>Optional. Can be used for a third round of sample trituration in the RNA extraction protocol.</td></tr><tr><td>1 ml syringe</td><td>VWR Scientific</td><td>BD309659</td><td>For very high value samples, a Luer-Lok syringe is recommended (&lt;i&gt;e.g.&lt;/i&gt;, VWR BD309628).</td></tr><tr><td>1-bromo-3-chloropentane (BCP)</td><td>Sigma</td><td>B9673</td><td></td></tr><tr><td>For 70% ethanol in DEPC water: 200 proof alcohol</td><td>Decon Laboratories, Inc.</td><td>+M18027161M</td><td>Mix 35 ml 200 proof alcohol + 15 ml DEPC water.&amp;nbsp;</td></tr><tr><td>For 70% ethanol in DEPC water: DEPC-treated water</td><td>Thermo Fisher Scientific</td><td>AM9922</td><td>Mix 35 ml 200 proof alcohol + 15 ml DEPC water.</td></tr><tr><td>RNA purification kit: PureLink RNA minikit</td><td>Thermo Fisher Scientific</td><td>12183018A</td><td>Final steps of RNA preparation.</td></tr><tr><td>DNase/Rnase-free water&amp;nbsp;</td><td>Gibco</td><td>10977</td><td>DEPC-treated water can also be used.</td></tr><tr><td>Spectrophotometer: Nanodrop 2000</td><td>Thermo Fisher Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Dnase I</td><td>Thermo Fisher Scientific</td><td>AM2222</td><td>Treat purified RNA to remove any DNA contamination before downstream appications.</td></tr><tr><td>Hydrophobic pen</td><td>Thermo Fisher Scientific</td><td>8899</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Gibco</td><td>14190</td><td>PBS for immunofluorescence protocol.</td></tr><tr><td>Donkey serum</td><td>Jackson ImmunoResearch Laoratories, Inc</td><td>017-000-121</td><td>Rehydrate normal donkey serum stock according to the manufacturer&#039;s instructions, then dilute an aliquot to 5% for immunofluorescence.&amp;nbsp; Normal goat serum can also be used.</td></tr><tr><td>eMHC antibody</td><td>University of Iowa Developmental Studies Hybridoma Bank</td><td>F1.652</td><td></td></tr><tr><td>Collagen VI antibody</td><td>Fitzgerald Industries</td><td>#70R-CR009x</td><td></td></tr><tr><td>Donkey anti-rabbit AlexaFluor488</td><td>Thermo Fisher Scientific</td><td>A21206</td><td></td></tr><tr><td>Goat anti-mouse IgG1 AlexaFluor546</td><td>Thermo Fisher Scientific</td><td>A21123</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Aqueous mounting media: Permafluor</td><td>Thermo Fisher Scientific</td><td>TA-030-FM</td><td>Only use mounting media designed for fluorescent applications with anti-fade properties.</td></tr><tr><td>Glass coverslip</td><td>VWR Scientific</td><td>16004-314</td><td>Use for mounting slides at the end of immunofluorescence protocl</td></tr><tr><td>Image analysis software: ImageProExpress</td><td>Media Cybernetics, Inc.</td><td>Image-Pro Express, or more advanced products</td><td>Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process; &lt;em&gt;e.g.&lt;/em&gt;, ImageProPremier.</td></tr><tr><td>Merge and map section images: Photoshop</td><td>Adobe</td><td>Photoshop</td><td></td></tr><tr><td>Cardiotoxin</td><td>Sigma</td><td>C9659</td><td>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><td>reverse transcription kit: Superscript III First-strand synthesis system</td><td>Thermo Fisher Scientific</td><td>18080051</td><td>Any validated, high quality reverse transcription reagents can be used.</td></tr><tr><td>Standard PCR: GoTaq Flexi polymerase system</td><td>Promega</td><td>M8298</td><td>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><td>SYBR green</td><td>Thermo Fisher Scientific</td><td>S7585</td><td>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><td>ROX: SuperROX, 15 mM</td><td>BioResearch Technologies, Inc. Novato CA</td><td>SR-1000-10</td><td>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><td>Real-time PCR</td><td>Applied Biosystems</td><td>7900HT</td><td></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。如表1所示，A260 / 280比率肌肉冷冻切片的RNA通常这些代表性样品在接近2或更高。为"纯"的RNA被认为具有A260 / 2.0和A260 280 / 1.8 280 2.2，在冷冻切片RNA样品的纯度是优异9。总RNA回收率为典型OVEř每用0.18收率样品10微克到超过1微克每起组织，对于最下游的应用提供足够的材料的毫克总RNA。值得注意的是，RNA浓度，提取总RNA，并自TA肌肉3天毒素损伤后每起始mg组织的RNA产量比从注射盐水的助教显著更高。这表明，有提高均质化时肌肉结构被广泛伤害和/或有第3天再生肌肉增加基因转录和/或RNA稳定破坏。 RNA的质量的持续存在是由简单的1×TAE / 1微升肌肉冷冻切片的RNA的1.5％琼脂糖凝胶电泳评估样品储存在-20℃下18个月后。突出18S和28S rRNA的频带依然在证明高RNA的质量的样品明显看出，即使在不理想的贮存条件下（ 图1A）。 肌肉冷冻切片RNA支持下游应用每个汇集冷冻切片样品的RNA的1微克用DNA酶处理的和从寡聚dT引物反转录。继核糖核酸酶处理，转录基因反向的总体积为30微升。用过量的扩增简单的非定量PCR运行，以确认该cDNA的生存能力。生肌调节因子4（MRF4），与肌肉分化上调的转录因子，采用先前报道的鼠标引物，有义:5&#39;-CTACATTGAGCGTCTACAGGACC和反义5&#39;-CTGAAGACTGCTGGAGGCTG 10放大，从模板2微升使用标准PCR。有来自左和右的TA cDNA样品的234 bp的MRF4片段的健壮的，特异性扩增，但不RT-（RNA包括，但没有反转录酶），RT DDH 2 O的（反向无RNA模板转录），或双蒸水2 O PCR控制（ 图1B）。相同的样品，一式三份地运行MRF4和mOaz1参考对照使用相对放大和被动荧光基准的实时PCR系统上4定量。 MRF4于在毒素注射右TA 0.097倍相比，注射盐水的左TA，通过ΔΔCT方法4计算表示。这类似于毒素损伤后3天低表达MRF4以前的报告，由于成熟的肌纤维11的损失。比较冷冻切片的RNA的定量PCR的一致性，Ct值被用于mOaz1参照基因进行比较。从六个样品，用17.242±1.483的SD的平均值的Ct检测mOaz1转录，而平均的Ct为36.332±3.61 SD RT-对照样品中（N = 4）。 mOaz1的Ct信号的整个样本的紧密分组表明，RNA的TA肌肉冰冻切片分离成预期下游RNA表达分析执行。 来自同窝对照小鼠为7μm胫骨前肌节毒素注射后第3天的间接免疫荧光染色的例子示以检测再生纤维（ 图2）。两部分从肌肉小于150μm从该区域以4.5的深度从近端肌肉表面被用于RNA分析，以4.6毫米收集。胚胎肌球蛋白重链（eMH​​C，红色）检测再生纤维，胶原蛋白VI（ColVI，绿色）概述了肌肉纤维，和DAPI污渍蓝色晶核，根据协议，4地区用浓核浸润（蓝色信号）表明毒素损伤部位通过eMHC阳性新近再生肌细胞的活化为明显。全断面图这样的例子是通过计算embryoni的比例用于再生的量化Ç肌球蛋白重链表达的纤维（红，新再生）或中央核纤维如先前报道1。值得注意的是，没有在图2A中的TA肌肉出奇损伤小。虽然有注射之间变化，典型毒素注射影响肌肉隔室1的一个更大的比例， 如图2B所示 。因此，该组织学分析表明，在图2A中的毒素损伤是最小的，并提供了从邻接肌肉冷冻切片RNA样品解释基因表达数据的重要工具。如果整个肌肉已经通过标准研磨方法用于RNA制备，该样品的意外小损伤区将使将歪斜下游分析离群值。相反，来自相同肌肉配对组织学定量允许从相邻部分的损伤的程度的直接测量。这使得能够使用inclu的长度/排除标准，以确保包括在下游的RNA所有样品分析满足最小损伤阈值或RNA的正常化分析根据损伤的大小。 <img alt="图1" src="/files/ftp_upload/55058/55058fig1.jpg" /> 图1:集资肌肉冰冻切片RNA的质量评估。 A）18S和28S核糖体RNA带已汇集肌肉冰冻切片的RNA制备18个月后的RNA突出。 b）非定量PCR MRF4以下反转录。的DNA梯的200和300bp的条带表示。 RT-，逆转与RNA模板，但没有逆转录酶转录反应。 RT-H 2 O，逆转录与DDH 2 O的，没有RNA模板。 的 H 2 0，用的DDH 2 O无模板PCR对照在这些实验中，毒素注射到右胫骨前（RTA）和盐水わ小号注入左侧（LTA）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55058/55058fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55058/55058fig2.jpg" /> 图2:样品组织学地图肌肉再生研究。 A，B）的单胫骨前肌节编译地图显示较差（A的例子毒素注射）和正常（B）毒素引起的损伤后3天。每个部分地图的白盒装区域被示出为插入图像，更高的放大倍率观察，红，胚胎肌球蛋白重链;绿色，胶原蛋白VI的细胞外基质蛋白;蓝色，DAPI核染色。比例尺，100微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55058/55058fig2large.jpg" target="_blank">请点击此处查看该图的放大版本</a>。 <img alt="表格1" src="/files/ftp_upload/55058/55058table1.jpg" /> 表1: 汇集肌肉冰冻切片代表RNA的产量和纯度测量。十六个胫骨前肌（TA）肌肉切片并汇集冷冻切片样本进行处理的RNA。纯化的RNA（1微升）用nanospectrophotometer进行分析。列统计信息是在电子表格中进行，利用统计软件进行组间比较。

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

Nanyang Technological University

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

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

科研

神经科学

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

发育生物学

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

医学

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

多鼠脑的同时 Cryosectioning

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

将小鼠大脑显微切割成功能和解剖学上不同的区域

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

生物学

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

骨骼肌中脂质液滴含量的纤维类型和亚细胞特异性分析

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

可变长度小鼠肌肉纤维的酶促分离和表征

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

作者聚焦：从小鼠后肢肌肉中分离长肌肉纤维，用于研究跨纤维类型的兴奋-收缩耦合

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

通过酶解离小鼠六块后肢肌肉获得的完整短、中和长骨骼肌纤维：超越趾短屈肌

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...