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 border="0" cellpadding="0" cellspacing="0" width="916">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:121px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:460px;">Comments/Description</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">4% Paraformaldehyde in phosphate buffered saline</td>
			<td style="width:121px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19202</td>
			<td style="width:460px;">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 &#176;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 height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S0389</td>
			<td style="width:460px;">Used for cryoprotecting tissue before freezing. Sucrose from any supplier may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O.C.T. compound&#160;</td>
			<td style="width:121px;">Tissue-Tek</td>
			<td style="width:119px;">4583</td>
			<td>Embedding medium for cryosectioning.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cryostat</td>
			<td style="width:121px;">Leica</td>
			<td style="width:119px;">CM 3050S</td>
			<td style="width:460px;">A Leica CM3050S cryostat was used for the preparations described here. Any cryostat capable of cutting 10 &#956;m sections may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass slides, 25 x 75 x 1 mm</td>
			<td style="width:121px;">Fisher</td>
			<td style="width:119px;">12-552-3</td>
			<td>Slides from any supplier may be used.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Gelatin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">G-9391</td>
			<td style="width:460px;">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 &#176;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 height="84">
			<td height="84" style="height:84px;width:216px;">Chromium(III) potassium sulfate dodecahydrate (chrome alum)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">243361</td>
			<td style="width:460px;">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 height="42">
			<td height="42" style="height:42px;width:216px;">Vectabond tissue adhesion reagent</td>
			<td style="width:121px;">Vector Labs</td>
			<td style="width:119px;">SP-1800</td>
			<td style="width:460px;">Optional substrate for better tissue adhesion to glass slides; gellatin-coated slides may be used instead.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween20</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9416</td>
			<td style="width:460px;">A detergent used to permeabilize tissue. Tween20 from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X100</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td style="width:460px;">A detergent used to permeabilize tissue. Triton X100 from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TACS 2 TdT fluorescein in situ apoptosis detection kit</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>Commercial kit for fluorescence-based TUNEL labeling.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase/nuclease</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>(included with kit)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase/nuclease buffer</td>
			<td style="width:121px;">Trevigen</td>
			<td style="width:119px;">4812-30-K</td>
			<td>(included with kit)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x phosphate buffered saline (PBS), pH 7.4</td>
			<td style="width:121px;">Amresco</td>
			<td style="width:119px;">780</td>
			<td style="width:460px;">Make 1x PBS for washes and dilutions. PBS from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DNase-free water</td>
			<td style="width:121px;">Quality Biologicals</td>
			<td style="width:119px;">351-029-131</td>
			<td>Water from any supplier may be used.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hoechst 33258</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">94403</td>
			<td style="width:460px;">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 height="42">
			<td height="42" style="height:42px;width:216px;">Parafilm M</td>
			<td style="width:121px;">multiple</td>
			<td style="width:119px;">807</td>
			<td style="width:460px;">Any other hydrophobic film or cover slip may be used. Available from multiple suppliers.&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent microscope with digital camera</td>
			<td style="width:121px;">&#160;--</td>
			<td style="width:119px;">&#160;--</td>
			<td style="width:460px;">Any fluorescent microscope capable of digitally capturing red, green, and blue fluorescence in separate channels may be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vectashield antifade media</td>
			<td style="width:121px;">Vector Labs</td>
			<td style="width:119px;">H-1000</td>
			<td>Antifade media from any supplier may be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass coverslips, No.1 thickness</td>
			<td style="width:121px;">Brain Research Labs</td>
			<td style="width:119px;">2222-1</td>
			<td style="width:460px;">Cover slips from any supplier may be used. The smallest size of 22x22 mm is sufficient for neonatal mouse leg sections.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nail polish</td>
			<td style="width:121px;">Ted Pella</td>
			<td style="width:119px;">114-8</td>
			<td style="width:460px;">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.&#160;</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 ...

detection-analysis-dna-damage-mouse-skeletal-muscle-situ-using-tunel

Research

JoVE Journal

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 vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

在胸腺的鼠标采用双光子显微镜的原位成像

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

对大鼠的过程<em在原位</em骨骼肌收缩性能

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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

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

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

从小鼠骨骼肌高通量微孔板呼吸测量最低数量的隔离线粒体

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

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.