Name: detection and analysis of dna damage in mouse skeletal muscle in situ using the tunel method
Uploaded: 2014-12-16T14:00:00.000+00:00
Duration: 9 min 54 s
Description: Watch this Scientific Journal Video about Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method at JoVE.com

This work was supported by NIH-NINDS grant RO1-NS065895 and NIH-NINDS grant 5-F31-NS076250-02. 
We thank JHU SOM Microscope Facility for the use of the cryostat.

注:在本协议中描述的所有动物的程序进行按照指南中的生26全国学院的实验室动物的护理和使用的建议。协议（MO13M391）是经美国约翰霍普金斯大学动物护理和使用委员会。 1.新生小鼠牺牲，解剖和固定<ol><li>牺牲一个新生小鼠通过CO 2吸入。 </li><li>立即切断膝盖以上的后肢。直到大约出生后第7天，腿部骨头都软得足以切断通过与大型实验室剪刀或锋利的刀片，并随后节低温恒温器。 </li><li>放置在磷酸盐缓冲盐水（PBS）在10毫升冰冷的4％多聚甲醛的后肢在一个15毫升管。 </li><li>浸泡修复多聚甲醛的后肢为4-24小时，在4℃。胚胎组织只需要几个小时固定用S的。 <ol><li>避免固定时间超过24小时，因为这可能会降低TUNE​​L法效率。 </li></ol></li><li>通过浸渍在蔗糖溶液Cryoprotect后肢。 <ol><li>在相同的15ml试管改变多聚甲醛在PBS中的10％蔗糖，孵育过夜，在4℃。 </li><li>变化到在PBS中30％蔗糖，孵育过夜，在4℃或直到后肢汇到管的底部。组织可能会留在30％蔗糖的时间较长，但前提是蔗糖已无菌过滤。 </li></ol></li></ol> 2.组织包埋<ol><li>填写标记嵌入模具中途与包埋剂。 </li><li>放置后肢上的包埋介质的顶部上，与切侧尽可能靠近到一个壁的模具。标记在模具的外侧后肢的方向。 <ol><li>嵌入多个后肢切在同一个块。但是，必须确保所有欣是非常重要的在块dlimbs被定向为平行于彼此，以获得部分在沿着肢体的轴相匹配的水平。 </li></ol></li><li>覆盖后肢与包埋剂。如果后肢转移而浇筑中，重新定位他们钝尖镊子或牙签。 </li><li>允许嵌入介质渗透到组织，在室温下至少15分钟。 </li><li>地方模具直接在干冰或异戊烷干冰冷冻包埋剂。一定要保持模具的水平在任何时候，避免包埋组织移位。 </li><li>保持冷冻的模具在-80℃，直到准备切割，长达数月。 </li></ol> 3. Cryosectioning嵌入式四肢<ol><li>设置低温恒温室温度至-20℃，对象温度至-18℃;调整目标温度需要下降。 </li><li>平衡的低温恒温室内部的组织块至少30分钟。正确的块温度erature是部分质量是至关重要的。 </li><li>切横截面的厚度为10微米或更小。较厚的部分不会被TUNEL试剂充分渗透。使用防卷板，叶片和叶片角度（一般为5°）推荐用于低温恒温器。 </li><li>收集的部分上明胶或vectabond涂覆载玻片在室温下。立即放入干冰幻灯片框。请用干冰放置在靠近低温恒温器近在咫尺单独的容器幻灯片框。 <ol><li>通过肢体的长轴切割横向连续切片，跳过100-200微米。收集至少3个相邻段在每一个串行水平。 </li></ol></li></ol> 4. TUNEL法对后肢部分使用市售的试剂盒（所有步骤在室温下，除非另有说明） <ol><li>解冻幻灯片使用节至室温并干燥过夜或过周末。 </li><li>补充水分通过immersin节克载玻片在PBS中用于在科普林缸2×10分钟。用干擦实验室，周围的部分尽可能删除尽可能多的液体。 </li><li>通透节<ol><li>使用一个非蛋白水解，皂甙类的商业试剂或0.5％吐温20和0.05％的Triton X100的PBS。 </li><li>将滑动到一个湿室中。一个简单的湿度室是一个具有紧盖子空枪头盒，具有加入刚好足够的水来覆盖盒子的底部。 </li><li>吸管足够试剂到滑动覆盖部分（50微升足以用于新生小鼠肢体）。不直接吸取到的部分中，作为重复的搅拌可导致部抬离滑动。 </li><li>使用钝头镊子，放置封口膜的小矩形上的部分，以摊开试剂的滴的顶部，并完全覆盖部分。如果有气泡的封口膜下形成，轻轻抬离，干燥，重新申请。 </li><li>孵育的幻灯片与permeabili矩阵特殊积试剂1小时在有盖的湿度室中。如果部分开始从滑动升空，通透时间可减少到30分钟。 </li><li>在一个科普林缸2×5分钟在PBS洗幻灯片。封口膜矩形应该浮到顶部并随后可以取出并丢弃。 </li><li>使用实验室擦拭，干周围的部分尽可能多的液体。 </li></ol></li><li>末端转移酶介导的DNA断裂标记<ol><li>按照试剂盒说明书进行1X末端转移酶标记缓冲液，吸管足够的标记缓冲到幻灯片，以支付组织切片（50微升足以新生小鼠四肢）。孵育在湿度室中5分钟。另外，沉浸在幻灯片中末端转移酶标记缓冲液中科普林罐子。 </li><li>按照试剂盒说明书进行末端转移酶标记组合。该套件包括钴，镁和锰的阳离子;锰阳离子行之有效上的各种组织，包括骨骼肌，神经系统TISS的UE，肝脏，皮肤，和骨。 </li><li>对于阳性对照，DNA酶（试剂盒中标记为"核酸酶"）添加到转移酶标记组合。可替代地，在37℃下预治疗在核酸酶缓冲液用DNase阳性对照部分1小时，按试剂盒的说明。保持DNA酶溶液和DNA酶处理的部分远离其他的实验部分，以防止交叉污染。 </li><li>对于阴性对照，省略标签组合转移酶的酶。 </li><li>使用实验室擦拭从幻灯片尽可能消除尽可能多的末端转移酶标记缓冲液。吸管末端转移酶标签组合到滑动（每节50微升），盖用封口膜的一个新的矩形，并孵育在湿度室中在37℃下1小时。 </li><li>按照试剂盒说明书进行1个挡车器。使用实验室擦拭以从滑动除去末端转移酶标记组合，并加入200-300微升终止缓冲液，以覆盖组织切片，并孵育在湿度室中5分钟。阿尔<html>替换地，沉浸在幻灯片中挡车器在科普林罐子。 </li><li>在PBS洗幻灯片2×2分钟。使用实验室擦拭从幻灯片尽可能消除尽可能多的PBS。 </li></ol></li><li>荧光标记<ol><li>按照试剂盒说明书准备荧光标签解决方案的DNA断裂标记的dNTP。吸管溶液到滑动（每节50微升），盖用封口膜的一个新的矩形，并孵育在湿度室中20分钟。 </li><li>洗幻灯片在PBS 2分钟的科普林罐子。使用实验室擦拭以从滑动除去尽可能多的PBS尽可能。 </li><li>稀释的Hoechst 33258核染色在PBS中的1微克/毫升和吸管到滑动（100-200微升足以覆盖部分）。孵育在湿度室中5分钟。 </li><li>洗3次5分钟，于PBS中。 </li><li>盖玻片与抗淬灭荧光安装媒体和密封用指甲油的边缘。 </li></ol></li></ol>jove_title"&gt; 5。数字图像采集<ol><li>获得使用常规荧光显微镜用DAPI，FITC和德克萨斯红滤波器（或等同物）的图像。使用较高的放大倍数的物镜（20X或40X），以验证该标记是成功的核（完整的或片段化）进行标记。 10倍的目标是足够的TUNEL阳性泪点定量。 </li><li>收集的Hoechst染色，TUNEL染色和自发荧光信号的图像作为三个独立的假色通道组合在一个图像。如果TUNEL荧光标签是绿色的红色滤光器，图像自身荧光信号，并且反之亦然钳住。 <ol><li>保留所有成像设置幻灯片后续阈值和定量之间的等价物。对于每一个过滤器，预先设定和记录的曝光时间，增益和分档（没有分级是最好的）;不使用自动曝光或增益设置。 </li><li>使用试错发现曝光和增益的设置，将最好的捕获INTEN的范围sities所有染色切片。确保没有饱和的像素获得的图像，因为这会偏向定量。 </li></ol></li><li>取的感兴趣的整个肌肉群的图像。这可能需要多个图像被"缝合"在一起。 </li></ol> 6.图像分析<ol><li>使用市售的图像分析程序来分析TUNEL染色数字图像。分析使用商业软件在此显示。可替代地，从美国国立卫生研究院的自由软件ImageJ的可用于分析TUNEL染色。 </li><li>毗邻染色与HE染色（H＆E）的TUNEL染色部分一个部分，并采取全断面数字图像与传统的明显微镜。结合使用该H＆E影像与解剖图谱5,18,19跟踪解剖结构进行量化。 </li><li>用TUNEL通道关闭时，手动跟踪的区域的轮廓进行量化，使用赫斯特和自体荧光渠道指导。所跟踪的区域转换成掩模（一组选定的象素的座标被分析）。 </li><li>打开TUNEL通道。使用的强度，在图像分析程序阈值函数，设置下限阈值，排除所有自发荧光信号。 </li><li>应用相同的阈值设置在书房集中的所有图像。阈值转换成选择口罩。 </li><li>对于每一个图像，结合肌区掩模和TUNEL掩模。新的组合将面膜仅代表追查肌肉区域内TUNEL信号。 </li><li>对合并面膜面膜进行定量分析，确定物体的数量和对象跟踪的肌肉区域内的TUNEL信号的区域。使用ImageJ的粒子分析仪功能，执行此。 </li></ol>

The authors have nothing to disclose.

的方法来检测和描述定量分析DNA损伤相关的细胞凋亡的小鼠骨骼肌。该过程包括组织收获，TUNEL染色，数字图像采集和图像分析。需要常见的组织学用品，工具，以及一个特殊的商业的TUNEL试剂盒是必要的。所需的基本的大设备项目是一个低温恒温器，落射荧光显微镜用数字图像的能力，以及用于图像分析的计算机系统。 实验者应该意识到潜在的陷阱。组织自发荧光的荧光成?...

双和单链DNA断裂12,23末端脱氧核苷酸转移酶（TDT）缺口末端标记（TUNEL）是利用末端转移酶酶连接的dUTP 3的进程的结束。 TUNEL法检测细胞凋亡和DNA损伤的由Gavrieli 等人 1,12,24首次报道在20年前。它已经被评估，并在不同的组织筹备7,23,27,40优化。 TUNEL法已被用于研究局部缺血诱导的细胞死亡的神经元6,14,29和心肌43,44的，兴奋毒性神经元细胞死亡30,31，并作为在关节炎治疗39的生物标记物。它也被用作在各种人类癌症2,3,15,32,37,38,42的预后因子和肿瘤细胞标记物。 DNA损伤和细胞死亡检测的替代方法存在，但他们有技术挑战和警告。 Southern印迹可用于quantifýDNA损伤在整个组织裂解7,9-11，但这种方法不提供蜂窝级别分辨率和难以量化。彗星试验是需要从细胞中提取4,20,28,36保存细胞核的替代的基于细胞的方法。虽然彗星试验工作良好对培养分离的细胞，它是更难以从骨骼肌组织8,21准备完整的细胞核。如同Southern印迹，彗星试验不提供从全肌肉组织匀浆细胞类型特定的信息。另一个替代TUNEL法是使用抗单链DNA 25,33,41或针对参与DNA损伤应答和细胞死亡途径（ 如 p53基因，H2AX，和半胱氨酸蛋白酶）13,17,22,40蛋白免疫组织化学。这样的基于抗体的方法需要的抗体的透彻表征和优异的抗体特异性，以产生一个高的信号 - 背景比。即使在规格IFIC抗体存在，它们可能需要通过抗原修复程序34,35变性靶蛋白。正如我们在这里讨论，抗原修复肌肉组织会导致不可接受的高自发荧光。不像其他方法，TUNEL实现DNA损伤检测具有高信号和低背景，特异性好，可以用简单的阳性和阴性对照，良好的组织穿透，不需要抗原修复和细胞级的分辨率进行测试。此外，TUNEL法需要大约4小时，以完成的，而另一种方法通常需要过夜孵育。 我们研究骨骼肌细胞死亡的脊髓性肌萎缩症（SMA）10是由谢，李和他的同事16产生的小鼠模型。为了量化凋亡细胞中的肌，我们开发了组织制备，染色和定量的方法，其工作原理鲁棒跨不同skeleTAL肌肉群在小鼠不同发育时间点。我们用市售的TUNEL标记试剂盒和市售的图像分析软件。我们也成功地使用了TUNEL测定结合免疫荧光染色在脊髓10。 此处所描述的方法是谁想要以评估骨骼肌组织病理学，疾病的机制，老化机制，和发育（前和产后）细胞死亡的调查是有用的。的TUNEL技术是DNA损伤和修复和细胞死亡在模型系统中，其中的细胞的一个子集是受影响和细胞水平分辨率是必要的研究特别有用。 这部影片描述了解剖，组织处理，切片，与小鼠骨骼肌基于荧光的TUNEL标记。它还描述了半自动化的TUNEL信号定量的方法。

<table><tbody><tr><td>4% Paraformaldehyde in phosphate buffered saline</td><td>Electron Microscopy Sciences</td><td>19202</td><td>For procedures described here, 4% solution was prepared fresh from powder. Paraformaldehyde from any supplier may be used. Prepared formaldehyde solution should be stored at 4 &amp;deg;C and should not be used after its expiration date (up to several months). Paraformaldehyde is a carcinogen and a toxin by inhalation and skin contact. Please follow precautions specified in the MSDS when handling paraformaldehyde.</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td>Used for cryoprotecting tissue before freezing. Sucrose from any supplier may be used.</td></tr><tr><td>O.C.T. compound</td><td>Tissue-Tek</td><td>4583</td><td>Embedding medium for cryosectioning.</td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 3050S</td><td>A Leica CM3050S cryostat was used for the preparations described here. Any cryostat capable of cutting 10 &amp;mu;m sections may be used.</td></tr><tr><td>Glass slides, 25 x 75 x 1 mm</td><td>Fisher</td><td>12-552-3</td><td>Slides from any supplier may be used.</td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G-9391</td><td>Gelatin is used to promote tissue section adhesion to glass slides. To coat glass slides with gelatin, dissolve 2.75 g gelatin and 0.275 g chrome alum in 500 ml distilled water, warm to 60 &amp;deg;C, dip slides for several seconds, and let dry. Gelatin from any supplier may be used. Alternatively, gelatin-precoated slides may be purchased.</td></tr><tr><td>Chromium(III) potassium sulfate dodecahydrate (chrome alum)</td><td>Sigma</td><td>243361</td><td>Chrome alum is added to gelatin solution to promote tissue adhesion on glass slides. It is a possible carcinogen and a toxin by inhalation and skin contact. Please follow precautions specified in the MSDS when handling chrome alum.</td></tr><tr><td>Vectabond tissue adhesion reagent</td><td>Vector Labs</td><td>SP-1800</td><td>Optional substrate for better tissue adhesion to glass slides; gelatin-coated slides may be used instead.</td></tr><tr><td>Tween20</td><td>Sigma</td><td>P9416</td><td>A detergent used to permeabilize tissue. Tween20 from any supplier may be used.</td></tr><tr><td>Triton X100</td><td>Sigma</td><td>T8787</td><td>A detergent used to permeabilize tissue. Triton X100 from any supplier may be used.</td></tr><tr><td>TACS 2 TdT fluorescein &lt;em&gt;in situ&lt;/em&gt; apoptosis detection kit</td><td>Trevigen</td><td>4812-30-K</td><td>Commercial kit for fluorescence-based TUNEL labeling.</td></tr><tr><td>DNase/nuclease</td><td>Trevigen</td><td>4812-30-K</td><td>(included with kit)</td></tr><tr><td>DNase/nuclease buffer</td><td>Trevigen</td><td>4812-30-K</td><td>(included with kit)</td></tr><tr><td>10x phosphate buffered saline (PBS), pH 7.4</td><td>Amresco</td><td>780</td><td>Make 1x PBS for washes and dilutions. PBS from any supplier may be used.</td></tr><tr><td>DNase-free water</td><td>Quality Biologicals</td><td>351-029-131</td><td>Water from any supplier may be used.</td></tr><tr><td>Hoechst 33258</td><td>Sigma</td><td>94403</td><td>Nuclear dye. Any blue fluorescent nuclear dye may be used. As a DNA-binding dye, Hoechst is a suspected carcinogen and should be handled with protective equipment to minimize skin contact.</td></tr><tr><td>Parafilm M</td><td>multiple</td><td>807</td><td>Any other hydrophobic film or cover slip may be used. Available from multiple suppliers.</td></tr><tr><td>Fluorescent microscope with digital camera</td><td>&amp;nbsp;--</td><td>&amp;nbsp;--</td><td>Any fluorescent microscope capable of digitally capturing red, green, and blue fluorescence in separate channels may be used.</td></tr><tr><td>Vectashield antifade media</td><td>Vector Labs</td><td>H-1000</td><td>Antifade media from any supplier may be used.</td></tr><tr><td>glass coverslips, No.1 thickness</td><td>Brain Research Labs</td><td>2222-1</td><td>Cover slips from any supplier may be used. The smallest size of 22 x 22 mm is sufficient for neonatal mouse leg sections.</td></tr><tr><td>Nail polish</td><td>Ted Pella</td><td>114-8</td><td>Used to seal coverslips. Nail polish from any supplier (including regular retailers) may be used. Avoid using nail polish with color or additives that may reflect light during fluorescent imaging.</td></tr></tbody></table>

detection and analysis of dna damage in mouse skeletal muscle in situ using the tunel method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

有了成功的染色，TUNEL阳性信号应该是足够明亮的自体荧光通过设定强度阈值隔离开来。在低放大倍数TUNEL阳性对象中可能出现的骨骼肌（ 图1A），为亮不规则碎片。然而，在较高的放大倍率，与经典的细胞凋亡形态一些TUNEL阳性对象应观察，如果所涉及的细胞死亡的类型是细胞凋亡（ 图1B）。阳性对照（DNA酶加）应该具有丰富TUNEL阳性信号，均匀地分布在所有的组织中的?...

Watch this Scientific Journal Video about Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method at JoVE.com

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

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Biology

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

16.2K 次观看.  Johns Hopkins School of Medicine. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling. 

视频: Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

科研

生物工程

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

发育生物学

鉴定小鼠前期停滞卵母细胞中的 DNA 损伤

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

作者聚焦：了解哺乳动物卵母细胞和植入前胚胎的 DNA 损伤反应 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

检测小鼠卵母细胞中的DNA双链断裂

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

生物学

使用 CUT&#38;Tag 分析小鼠成肌细胞中的基因组组蛋白标记

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&amp;Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&amp;Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&amp;Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&amp;Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

作者聚焦：应对肌肉干细胞研究中的挑战和创新

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

在小鼠成肌细胞研究中使用靶标和标记下的切割（CUT&#38;Tag）测定

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile ...

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

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

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 ...

检测和DNA损伤分析小鼠骨骼肌

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此视频中的章节

在检测DNA损伤的体外半人工分子机器，其使用的大会

骨骼肌损伤诱导衰老和体内重编程的评价

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

MG53介导的细胞膜使用维修的可视化在体内在体外系统

在小鼠成肌细胞研究中使用靶标和标记下的切割（CUT&Tag）测定

在小鼠成肌细胞研究中使用靶标和标记下的切割（CUT&Tag）测定

在小鼠成肌细胞研究中使用靶标和标记下的切割（CUT&Tag）测定

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