Name: identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies
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Description: Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

我们感谢结缔组织国立研究院光成像部分提供显微镜和技术帮助。MF20 和 Pax7 抗体是从发展研究中获得的, 在发育研究院的主持下开发的杂交瘤银行, 由爱荷华州的爱荷华大学生物科学系维护。这项工作得到了国家卫生研究院结缔组织国立研究院的校内研究项目的支持。

本协议以成年小鼠 (2-6 月)、胫骨前 (TA) 和伸趾肌 (EDL) 为例, 进行免疫荧光染色。所有的步骤处理小鼠和肌肉组织解剖已被批准的动物护理和使用委员会 (ACUC) 的结缔组织国立研究院/NIH。
1. 从小鼠后肢解剖 TA/EDL 肌肉
<ol><li>弄死在 CO2填充的安乐死室中的鼠标在 NIH 的 ACUC 的指导下。如有需要, 执行颈椎脱位。</li>
	<li>将鼠标正面放在解剖垫上 (仰卧位)。将70% 乙醇喷洒到其腹部皮肤上。在鼠标腹部皮肤的中心水平上切开1-2 厘米的开口, 然后通过拉向鼠标脚的开口 deskin 鼠标。露出老鼠后肢的一侧。</li>
	<li>使用两个锋利的钳: 使用一把肌腱连接 ta/edl 到脚踝, 并使用另一个打破和剪切的筋膜覆盖 TA/edl。当筋膜脱落后, 使用镊子的锋利尖端, 通过在 ta/edl 肌肉下面运行尖端, 将 ta/edl 从胫骨骨中分离出来。</li>
	<li>用剪刀切断脚踝肌腱, 同时用镊子钳住。用剪刀沿从脚踝到膝盖的 ta/edl 的纵线修剪肌肉, 同时用镊子将 ta/edl 从脚踝一侧抬起。切开膝盖侧肌腱, 将整个 TA/EDL 从胫骨中分离出来。</li>
</ol>2. 对 TA/EDL 肌肉的低温嵌入
<ol><li>在容器中准备一个液氮浴, e. g, 一个液态氮气杜瓦瓶。把丁烷倒入一个小塑料烧杯 (大约20毫升), 半满。然后把烧杯放在液氮浴中冷冻。几分钟后, 检查丁烷是否冻结。 注: 液氮可以用一桶干冰代替, 但冻结时间较慢。</li>
	<li>将解剖过的 TA/EDL 放置在小塑料注射器柱塞的平坦表面上。充分覆盖 TA/EDL 与几滴最佳切削温度 (O.C.T.) 化合物 (冷冻介质)。</li>
	<li>把丁烷烧杯从液氮浴中取出, 用一个温暖的物体 (e. g) 解冻表面, 剪刀柄。快速将 O.C.T. 覆盖的 TA/EDL 放到融化的丁烷中, 将组织冻结。</li>
	<li>用镊子将嵌入的组织从柱塞中分离出来, 放入1.5 毫升离心管。在处理更多的样品时, 把管子放进液氮储存。 注: 在短期 (6 月) 内, 植入的组织可以长期保存在-80 摄氏度 (2 年) 或-20 摄氏度。</li>
</ol>3. 恒温器切片
<ol><li>将一滴 O.C.T. 化合物添加到恒温器标本持有者的中心。等到 O.C.T. 化合物几乎凝固。快速将嵌入的 TA/EDL 粘在 O.C.T. 化合物上, 垂直于脚踝侧向下和膝侧。</li>
	<li>将标本架装在恒温器上, 在10µm 间隔处切开组织, 在一张幻灯片上收集冰冻切片, 大约4-8 节。</li>
	<li>在室温下将部分干燥至少3小时至一夜。 注意: 虽然在冷冻室中干燥一夜或更长时间是可以接受的, 但它必然会增加自体荧光。为取得最佳效果, 请将切片干燥0.5 小时, 然后立即进行固定和染色步骤。</li>
	<li>收集幻灯片框中的幻灯片并立即使用它们, 或将幻灯片存储在-80 °c 或-20 °c 以供以后使用。</li>
</ol>4. 荧光染色试剂的制备
<ol><li>
准备缓冲区:
	<ol>
<li>从 10x pbs 中制备2升1x 磷酸盐缓冲盐水 (pbs)。</li>
		<li>在 1x PBS 中准备40毫升4% 多聚甲醛 (粉煤灰), 从16% 个粉煤灰。</li>
		<li>准备2升 PBST: 1x PBS 与 0.1% Triton-100。</li>
		<li>准备50毫升的 PBST: PBST 与2% 牛血清白蛋白 (BSA)。</li>
		<li>准备10毫升的 PBST: PBST 与 2% BSA 和5% 正常山羊血清。</li>
		<li>准备1毫升的阻断溶液。稀释 AffiniPure 片山羊抗鼠 IgG (H + L) (1:10) 在 PBST。</li>
		<li>准备200毫升的柠檬酸1x 缓冲液。 注意: 上面的解决方案卷足够至少5张幻灯片。卷应根据幻灯片的数量和幻灯片容器的大小进行调整。</li>
	</ol>
</li>
	<li>
准备抗体污渍:
	<ol>
<li>主要抗体 (工作浓度): 通过稀释 Pax7 单克隆小鼠抗体 (IgG1) (1-5 µg/毫升) + 层粘连蛋白多克隆兔抗体 (0.5-2 µg/毫升) + MF20 单克隆小鼠抗体 (IgG2b) (0.5-2.5 µg/毫升) 在 PBST。 注意: MF20 单克隆鼠标抗体 (IgG2b) 是可选的。</li>
		<li>二级抗体 (工作浓度): 通过稀释山羊抗小鼠 IgG1 交叉吸附二次抗体, 制备1.2 毫升二级抗体混合物, Alexa 氟 488 (0.5-2 µg/毫升) + 山羊抗兔 IgG (H + L) 高度交叉吸附二次抗体,alexa 氟加 555 (0.5-2 µg/毫升) + 山羊抗鼠 IgG2b交叉吸附二次抗体, Alexa 氟 647 (0.5-2 µg/毫升) 在 PBST b G。 注: Alexa 647 是可选的。</li>
	</ol>
</li>
</ol>5. 免疫荧光染色步骤
<ol><li>
水化用粉煤灰固定冷冻部分。
	<ol>
<li>从幻灯片框中删除一些 TA/EDL 冰冻部分幻灯片。在室温下, 在滑动夹盘中加热幻灯片。</li>
		<li>使用液体阻挡笔 (PAP 笔) 绘制一个区域, 其中包括幻灯片上的所有冰冻部分。这个圆圈将阻止解决方案从幻灯片上流出。</li>
		<li>把托盘带到实验室油烟机。在带圆圈区域内的幻灯片上添加300-500 µL 4% 粉煤灰, 以在室温下固定部分10分钟. 在滑动容器中, 至少 3x 5 分钟, 用 1x PBS 清洗粉煤灰。 注意: 粉煤灰是有毒的, 是有害的废物;应小心操作, 并将其弃置在危险废物容器内。</li>
	</ol>
</li>
	<li>
抗原检索
	<ol>
<li>用200毫升柠檬酸盐缓冲填充另一个滑动容器。</li>
		<li>将幻灯片转移到容器中。将容器与幻灯片转移到压力锅中, 在高压模式下将幻灯片煮10分钟。</li>
		<li>用排水10分钟冷却容器. 将所有幻灯片转移到具有 PBST (200 毫升) 的新容器中, 用 PBST 冲洗幻灯片至少 3x 5 分钟。</li>
	</ol>
</li>
	<li>
阻塞
	<ol>
<li>将水浸泡的 kimwipes 放在托盘上, 以创建潮湿的环境, 以避免幻灯片的干燥。</li>
		<li>将幻灯片移回托盘, 并在每张幻灯片上添加200µL 的阻塞解决方案。用瓶盖盖上托盘, 并让阻塞发展30分钟。 注: Pax7 抗体是一种小鼠单克隆抗体, 因此在小鼠的肌肉组织中有不特异的结合小鼠 IgG, 创造高背景。使用 AffiniPure 的碎片山羊抗鼠 IgG (H + L) 阻止鼠标对鼠标不特异绑定。</li>
	</ol>
</li>
	<li>
应用主要抗体。
	<ol>
<li>在幻灯片容器中用 PBST 冲洗幻灯片。然后将幻灯片移回染色托盘。</li>
		<li>在每张幻灯片上应用200µL 的主抗体组合, 用盖子覆盖托盘, 并让反应在室温下发展1小时或在4摄氏度过夜。 注意: 在4摄氏度的隔夜反应给出最好的结果, 尽管在室温下发展反应给出了令人满意的结果。肌球蛋白 MF20 抗体可以添加到混合, 以执行三重 (Pax7, 层粘连蛋白, MF20) 标签。MF20 污渍分化的肌肉纤维。这一步也作为另一个阻断步骤与 PBST, 以减少潜在的非特异性结合的第二抗体 (山羊抗体) 在以下步骤。</li>
	</ol>
</li>
	<li>
应用二级抗体。
	<ol>
<li>用 PBST 在室温下至少 3x 5 分钟内冲洗出主要抗体。</li>
		<li>添加200µL 的二次抗体混合到每张幻灯片, 并允许反应在室温下发展至少1小时。 注: 对于 Pax7+Laminin, 使用山羊抗鼠 IgG1 488 和山羊抗兔 alexa 555。 对于 Pax7+Laminin+MF20, 添加山羊抗鼠标 IgG2b Alexa 647 在混合。</li>
		<li>用 PBST 清洗二次抗体, 至少 3x 5min。</li>
	</ol>
</li>
	<li>
DAPI (4 ', 6-diamidino 2-苯基吲哚) 反着色和安装
	<ol>
<li>稀释 DAPI (1:20,000) 在 PBST 中的滑动容器中,例如, 10 µL DAPI 200 毫升的 PBST。</li>
		<li>将 DAPI 容器中的幻灯片着色3分钟。</li>
		<li>用 PBST 洗净 DAPI 至少 5x 5 分钟。</li>
		<li>装入带有安装介质的幻灯片。用盖玻片覆盖幻灯片。 注意: DAPI 有毒、危险;应小心处理, 并在危险废物瓶中处置。</li>
	</ol>
</li>
</ol>6. 荧光显微术
注: 上述免疫荧光染色协议将允许 SCs 可视化, 无论是广域荧光或共聚焦显微镜。确定 SCs 可能最初具有挑战性。下面是一些可以帮助您的建议步骤。
<ol><li>使用 DAPI 通道和低缩放目标可视化幻灯片上的部分。</li>
	<li>从低到高切换放大倍数。要观察 SCs, 至少需要20X 的目标)。</li>
	<li>使用 488 (FITC)/555 (罗丹明) 的双过滤器立方体同时观察 Pax7 和层粘连蛋白信号, 以识别 SCs。在这一背景下, Pax7 染色的信号与噪声有较好的对比。</li>
	<li>快速将通道从 FITC 切换到 DAPI, 以正确识别 SCs。所有的 SCs 都应该 Pax7/DAPI-positive。</li>
</ol>

<ol>
	<li>Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+and+cell+lineages+that+underlie+the+formation+of+skeletal+muscle.">Gene regulatory networks and cell lineages that underlie the formation of skeletal muscle.</a> Proc Natl Acad Sci U S A. 114 (23), 5830-5837 (2017).</li><li>Buckingham, M., Relaix, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX3+and+PAX7+as+upstream+regulators+of+myogenesis.">PAX3 and PAX7 as upstream regulators of myogenesis.</a> Semin Cell Dev Biol. 44, 115-125 (2015).</li><li>Relaix, F., Buckingham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+insect+eye+to+vertebrate+muscle:+redeployment+of+a+regulatory+network.">From insect eye to vertebrate muscle: redeployment of a regulatory network.</a> Genes Dev. 13 (24), 3171-3178 (1999).</li><li>Chang, N. C., Chevalier, F. P., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+Cells+in+Muscular+Dystrophy+-+Lost+in+Polarity.">Satellite Cells in Muscular Dystrophy - Lost in Polarity.</a> Trends Mol Med. 22 (6), 479-496 (2016).</li><li>Mauro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cell+of+skeletal+muscle+fibers.">Satellite cell of skeletal muscle fibers.</a> J Biophys Biochem Cytol. 9, 493-495 (1961).</li><li>Kuang, S., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+biology+of+satellite+cells+and+their+therapeutic+potential.">The emerging biology of satellite cells and their therapeutic potential.</a> Trends Mol Med. 14 (2), 82-91 (2008).</li><li>Wang, Y. X., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+cells,+the+engines+of+muscle+repair.">Satellite cells, the engines of muscle repair.</a> Nat Rev Mol Cell Biol. 13 (2), 127-133 (2011).</li><li>Rinaldi, F., Perlingeiro, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+skeletal+muscle+regeneration:+therapeutic+potential+and+roadblocks.">Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks.</a> Transl Res. 163 (4), 409-417 (2014).</li><li>Le Grand, F., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+satellite+cells+and+adult+myogenesis.">Skeletal muscle satellite cells and adult myogenesis.</a> Curr Opin Cell Biol. 19 (6), 628-633 (2007).</li><li>Giordani, L., Puri, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+control+of+skeletal+muscle+regeneration:+Integrating+genetic+determinants+and+environmental+changes.">Epigenetic control of skeletal muscle regeneration: Integrating genetic determinants and environmental changes.</a> FEBS J. 280 (17), 4014-4025 (2013).</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 (10), 1612-1624 (2015).</li><li>Feng, X., Adiarte, E. G., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+acts+directly+on+the+zebrafish+dermomyotome+to+promote+myogenic+differentiation.">Hedgehog acts directly on the zebrafish dermomyotome to promote myogenic differentiation.</a> Dev Biol. 300 (2), 736-746 (2006).</li><li>Juan, A. H., 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=Polycomb+EZH2+controls+self-renewal+and+safeguards+the+transcriptional+identity+of+skeletal+muscle+stem+cells.">Polycomb EZH2 controls self-renewal and safeguards the transcriptional identity of skeletal muscle stem cells.</a> Genes Dev. 25 (8), 789-794 (2011).</li><li>Jackson, K. A., Snyder, D. S., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber-specific+green+autofluorescence:+potential+for+stem+cell+engraftment+artifacts.">Skeletal muscle fiber-specific green autofluorescence: potential for stem cell engraftment artifacts.</a> Stem Cells. 22 (2), 180-187 (2004).</li><li>Lepper, C., Conway, S. J., Fan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+satellite+cells+and+embryonic+muscle+progenitors+have+distinct+genetic+requirements.">Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements.</a> Nature. 460 (7255), 627-631 (2009).</li></ol>

作者没有什么可透露的。

上述协议是基于对斑马鱼骨骼肌的 Pax7/MF20 染色方法12。使用的解决方案和阻止步骤是相同的或类似的。使用的抗体是相同的。调整后的步骤是基于小鼠肌肉组织和 SCs 的特点。首先, 在混合中加入层粘连蛋白抗体, 以帮助可视化和确认 SCs 的位置。在使用488/555 的双滤池时, 在显微镜下计算 SCs 的数量特别有用;这大大增强了 SC 衍生的 Pax7 信号, 脱颖而出, 从嘈杂的 autofluorescent 背景?...

骨骼肌由多核肌肉细胞组成, 称为 myotubes, 组织在肌纤维, 通过收缩产生力量和运动。大多数骨骼肌, 除了一些头面部肌肉, 都来源于一个叫做 somite1的临时胚胎结构。肌原细胞分层从上皮 somite 变成成细胞。成肌细胞进一步分化为 myotubes 形成多核肌纤维。上述过程称为肌, 其特点是由世俗调控的基因表达控制。肌质前体表达 Pax3 和 Pax7, 而成细胞表达 MyoD 和/或 Myf5 和肌细胞膜表达肌形成蛋白和球蛋白2, 3.肌肉生长是一个过程中, 肌纤维变得更大, 通过加入更多的 myonuclei 到现有的纤维 (增生) 和增加肌肉纤维大小 (肥厚) 4.在肌肉生长过程中, 有一个可持续的肌细胞来源, 它们具有干细胞的特性, 可以区分和自我更新。这些细胞被称为卫星细胞的基础上, 其物理位置之间的肌膜包裹 (细胞膜的肌纤维) 和基底叶片5。SCs 在青少年时期 (前2-3 周的产后小鼠) 大力促进肌肉生长, 但在休息的成年肌肉中成为静止的6。值得注意的是, 它们可以重新激活, 以应对肌肉损伤, 并分化为新的肌肉细胞, 以修复受损的肌肉7。
干细胞的性质使研究的 SCs 相关的基本肌肉生物学和治疗肌肉疾病的8。因此, 在过去几十年中, 它一直是一个激烈调查的领域。在剖析 SCs9、10的遗传学和表遗传学方面取得了巨大进展。在11的方式下开发并优化了用于隔离和识别 SCs就地的技术。免疫荧光染色可通过使用特定的抗体 (包括 Pax7) 识别 SCs。然而, 由于 SCs 的稀缺性和体积小, 结合了成人骨骼肌组织的强烈的自动荧光, 使可视化具有挑战性。在这里, 我们描述了一个免疫荧光染色协议优化的小鼠肌肉组织为 Pax7 和基于现有的方法, 斑马鱼肌肉12。此外, 用一个带有明显荧光的层粘连蛋白抗体来识别 SCs 所在的基底层。该议定书一贯允许在所有经过测试的生理条件和发育阶段 Pax7-positive SCs 和肌原体的可视化。

<table><tbody><tr><td>methylbutane</td><td>Sigma-Aldrich</td><td>M32631</td><td></td></tr><tr><td>Optimal Cutting Temperature (O.C.T.) compound</td><td>Electron Microscopy Sciences</td><td>62550-01</td><td></td></tr><tr><td>10x PBS</td><td>Gibco, Themo Fisher</td><td>70011-044</td><td></td></tr><tr><td>16% PFA</td><td>TED PELLA</td><td>50-00-0</td><td></td></tr><tr><td>Triton-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Thermo Fisher</td><td>0 1-6201</td><td></td></tr><tr><td>AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories Inc.</td><td>115-007-003</td><td></td></tr><tr><td>20x Citrate Buffer</td><td>Thermo Fisher</td><td>00 500</td><td></td></tr><tr><td>Pax7 mono-clonal mouse antibody (IgG1) (supernatant)</td><td>Developmental Study Hybridoma Bank</td><td>N/A</td><td></td></tr><tr><td>Laminin polyclonal rabbit antibody</td><td>Sigma-Aldrich</td><td>L9393</td><td></td></tr><tr><td>MF20 mono-clonal mouse antibody (IgG2&lt;sub&gt;b&lt;/sub&gt;) (supernatant)</td><td>Developmental Study Hybridoma Bank</td><td>N/A</td><td></td></tr><tr><td>Goat anti-Mouse IgG1 cross-absorbed secondary antibody, Alexa Fluor 488</td><td>Thermo Fisher</td><td>A-21121</td><td></td></tr><tr><td>Goat anti-Mouse IgG2&lt;sub&gt;b&lt;/sub&gt; cross-absorbed secondary antibody, Alexa Fluor 647</td><td>Thermo Fisher</td><td>A-21242</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555</td><td>Thermo Fisher</td><td>A32732</td><td></td></tr><tr><td>Leica CM1860 cryostat</td><td></td><td></td><td></td></tr><tr><td>Leica DM6000 wide-field fluorescent microscope</td><td></td><td></td><td></td></tr><tr><td>Leica DMR wide-field fluorescent microscope</td><td></td><td></td><td></td></tr><tr><td>Zeiss LSM510 confocal microscope</td><td></td><td></td><td></td></tr><tr><td>Zeiss LSM780 confocal microscope</td><td></td><td></td><td></td></tr><tr><td>Cuisinart electronic pressure cooker</td><td></td><td></td><td></td></tr></tbody></table>

identification of skeletal muscle satellite cells by immunofluorescence with pax7 and laminin antibodies

免疫荧光是一种有效的方法, 有助于识别不同的细胞类型的组织切片。为了研究所需的细胞数量, 在组织切片上应用特定细胞标记的抗体。在成年骨骼肌中, 卫星细胞 (SCs) 是促进肌肉修复和再生的干细胞。因此, 在不同的生理条件下, 对卫星细胞种群进行可视化和跟踪是十分重要的。在静止的骨骼肌中, SCs 位于基底叶片和肌纤维膜之间。在肌纤维或细胞培养中, 用于识别 SCs 的常用标记是配对盒蛋白 Pax7。本文提出了一种优化 Pax7 免疫荧光协议的骨骼肌切片, 最大限度地减少非特异染色和背景。另外还添加了一种识别基底叶片蛋白质 (层粘连蛋白) 的抗体, 以帮助识别 SCs. 类似的协议也可以用来执行双重或三重标记与 Pax7 和抗体的额外蛋白质的兴趣。

按照上述步骤, 可以成功地将 SCs 可视化为成人休息肌肉部分的荧光显微镜 (图 1)。虽然成人肌肉组织有强的自动荧光在某些类型的纤维, 明亮的 Alexa 系列染料能克服背景噪声和信号突出 (图 1A, B; 箭头头)。双光子共焦显微镜捕获相对较清洁的图像 (图 1B) 比宽场荧光显微镜 (图 1A...

Watch this Scientific Journal Video about Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies at JoVE.com

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

卫星细胞的精确识别对于研究其在各种生理和病理条件下的功能至关重要。本文提出了一种基于免疫荧光染色的成人骨骼肌切片卫星细胞识别的协议。

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

identification-skeletal-muscle-satellite-cells-immunofluorescence

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20.9K 次观看.  National Institutes of Health. 卫星细胞的精确识别对于研究其在各种生理和病理条件下的功能至关重要。本文提出了一种基于免疫荧光染色的成人骨骼肌切片卫星细胞识别的协议。

视频: Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

遗传学

从小鼠肢体肌肉中分离卫星细胞和流式细胞术分析

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

作者聚焦:小鼠肌肉干细胞中的 Cistrome 分析 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

一种用于FACS分离的小鼠卫星细胞的CUT&amp;RUN分析的有效方案

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

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

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

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

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

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

生物学

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

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此视频中的章节

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

隔离和卫星细胞的表征鼠头Branchiomeric肌肉

从小鼠骨骼肌分离I型和II型周细胞

通过心肌毒素损伤和在 Vivo 中注射自传递 siRNA 在骨骼肌再生过程中的卫星细胞功能分析

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

一种用于FACS分离的小鼠卫星细胞的CUT&RUN分析的有效方案

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

分离，培养和移植肌卫星细胞

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备