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 (IgG2b) (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 IgG2b 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

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Developmental Biology

Unit 1

Extracellular Matrix in Animals

SCIENTISTS IN ACTION

20.8K 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 ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

制备及文化活动从成年和老年人类骨骼肌成肌前体细胞/成肌细胞主要的

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...