The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.
骨骼肌是因为它的结构和功能,这需要用于组织收集特定的协议来从功能性的,细胞,分子和病理评估获得最佳结果的唯一组织。由于见于先天性肌肉疾病以及潜在的供固定干预承认这些特征的一些病理异常的微妙,当评估骨骼肌先天性肌肉疾病冷冻肌肉病理评价,优选固定的肌肉。此外,为了产生严重的冰冻文物肌肉的潜在需要特定的预防措施,冻结骨骼肌冻结其他组织时并不常用组织学检查时。此手稿描述用液氮冷却以保持最佳的骨骼肌形态的协议为使用异戊烷(2 – 甲基丁烷)骨骼肌的快速冷冻。这个过程也有效于Freezing组织用于基因或蛋白表达的研究。此外,我们还整合了冷冻协议到一个更广泛的过程,还介绍了组织的短期分流首选方法(1)单根纤维的功能性研究及(2)成肌细胞培养,重点是收集必要的最低限度的努力组织并运输到专门的研究或参考实验室来完成这些研究。总体而言,这份手稿提供了如何新鲜组织可以有效地分布于各种表型研究的概况,从而为相关的先天性肌肉疾病的病理研究提供了标准操作规程(SOP)。
Skeletal muscle is a unique tissue because of its structure and function, which can present a number of challenges when evaluating its pathology. While it is sufficient, and often even optimal, to evaluate most tissues following formalin fixation and paraffin embedding, this process leads to the production of cell shrinkage artifacts that impair the evaluation of critical phenotypes in congenital muscle disease (including myofiber size and shape). Additionally, frozen muscle is required when performing many histochemical stains (including the reduced nicotinamide adenine dinucleotide (NADH), cytochrome oxidase (COX), succinate dehyrogenase (SDH), adenosine triphosphatase (ATPase), and Oil Red O stains) that are essential for the detection of specific pathological abnormalities required to diagnose a variety of congenital myopathies, mitochondrial myopathies, and storage diseases. Many antibodies and molecular tools used to characterize muscle morphology and function have consequently been developed and optimized only for frozen tissue, which further limits the usefulness of fixed tissue preparations in studies of skeletal muscle biology. As a result, it has become standard practice to freeze skeletal muscle specimens for most clinical and research indications in the field of congenital muscle disease, despite the technical challenges that appropriate freezing might pose.
The freezing of skeletal muscle can be technically challenging due to the high likelihood of ice crystal formation within myofibers when an inappropriate freezing protocol is followed1. In most cases, freezing artifacts are encountered when the water content of the tissue is too high or when the freezing process is too slow. The water content of tissue can be accidentally increased by immersion in fluid, such as transport of the muscle in saline; or freezing can be compromised if the tissue has been entirely immersed in OCT. Both of these procedures can lead to catastrophic freezing artifacts in muscle tissue. Additionally, a number of small missteps in the freezing process may cause slow freezing or accidental thawing of samples after appropriate freezing, and these issues can similarly complicate pathological evaluation.
As new models and treatments for skeletal muscle disease are developed, it will become increasingly useful to use standard operating procedures (SOPs) to evaluate skeletal muscle phenotypes and treatment efficacy. This is particularly important for the pathological evaluation of congenital muscle diseases, as the recognition of subtle phenotypes seen in many of these disorders can be impaired by improper tissue preparation. This report represents an effort by a Congenital Muscle Disease Consortium to establish a skeletal muscle freezing SOP for the study of congenital muscle disease models.
The protocol presented here describes a strategy used in our clinical and research labs to optimize assessment of frozen muscle pathology, while also being suitable for a variety of protein-based and genetic studies. Specific strategies to prepare skeletal muscle specimens for electron microscopy are also presented. Additionally, as single fiber functional testing of frozen tissue is possible2,3, experts in these techniques have provided details on optimal tissue handling protocols. Unfortunately, the isolation of myoblast cell lines cannot be effectively performed on pre-frozen tissue, so a strategy is presented herein for the short-term storage and transport of muscle tissue for myoblast culture establishment at outside laboratories. Overall, the methods described here provide guidelines for the appropriate processing of muscle tissue for a variety of pathological, functional, molecular and cellular studies, regardless of the on-site capabilities of a given laboratory.
骨骼肌是一个结构和功能上独特的组织,并专门编写的程序是必要的,以允许的结构和功能参数的最佳评估。而各种组织中通常冻结在临床和研究上下文病理研究,对于非肌肉组织冷冻协议冷冻前通常涉及组织在OCT中的全浸没。如示于图3中 ,这样的协议不适合于骨骼肌的病理评价,但足以类似于这里所描述的协议,这是一个经常遇到的错误。本文的目标是提供一个简单的协议,用于肌肉的妥善处理,以避免这样的问题。建议也被编上肌肉异地生理和细胞研究的妥善处理,努力促进收购的高质量的数据在案件以外的核心实验室磨片重新现场研究是不可取的或可能的。
作为在该协议指出,这是在肌肉的正确处理绝对必不可少的病理研究内容包括最小化的组织中的水含量,从而降低在该肌肉被冻结的温度,并增加在该冷冻达到的速度。组织或冷冻过程(由温度不足或缺乏的冷冻剂和所述组织之间的直接接触,产生如遇到用液氮)的过度缓慢内过多的水分会导致冻结的工件,可以削弱病理分析。华侨城提供了组织液的额外来源,许多实验室使用其他粘合剂像西黄蓍胶作为嵌入基板。即使在被适当地执行冻结的情况下,应注意通过与RT容器或器具接触,以避免意外后解冻标本。因而,一个成功的冻结过程需要一定程度的规划,其中包括一个预冷所使用的所有仪器和容器。当遇到冷冻的工件,有这里描述的是足以满足大多数病理学评估的组织的恢复方法。然而,这种冷冻/解冻循环不提供完美的组织学和有损害的组织(除了需要重新冻结的组织中的时间)的其它分子或酶研究的电位,因此,使用适当的初始冷冻做法是大大优选。在冷冻工件被预先冷冻的组织中遇到的情况下,然而,这里描述的技术可以是非常有用的。
固定组织的EM可以提供,此前组织收集需要规划具体的技术挑战和处理。采集标本的EM时最常见的错误包括使用组织碎片太厚的glutaraldehyd的e来渗透。戊二醛只从一个给定的表面,护理穿透约0.1厘米进入肌肉组织,应采取措施,确保了EM的样本中的一维是厚度不超过0.2厘米。此外,由于EM是直接评估收缩装置的极好手段,一些研究者已经开发战略,以预拉伸或预拉伸前的肌肉固定,使收缩元素的测量在生理相关的张力。没有标准协议,用于预张紧,但两种策略在本协议简要介绍。应当指出,试图预张紧的肌肉会产生不可预测的结果,除非它们被做在一个非常特定的方式,并且它可能是优选的修复肌肉松驰状态,以防止在肌小节长度伪迹的变化通过一个非标准预张紧步骤8,9。对于肌肉在这种特定的测量是没有必要的(包括第二个白用于临床目的进行iopsies),努力以预张力的肌肉一般不进行,并且对肌肉形态的主要效果是肌节的肌非均匀间距。
本文代表了第一个系列提供标准操作程序进行测试的先天性肌肉疾病领域的表现,它代表了20位专家在执行例行的细胞,分子,功能性,生理性的先天性肌肉疾病领域的努力,病理研究。发表的SOP的范围内将提供在未来的一年,并为每个必要的协议和相应的出版形式,这在先天性肌肉疾病协会研讨会于2013年四月在华盛顿举行讨论这个SOP努力的目标是提供路线图进行必要的测试和样本分析中的先天性肌肉疾病领域1)标准化在我们的领域作为米的做法和终点UCH越好,2)提供指导标准的做法,新的研究人员在我们的领域。我们相信,这些资源将促进新的调查进入我们的下深入研究的领域,从而提高了可进行的研究范围之内。此外,实践标准化将是比较不同的研究数据,并在规划和实施临床前和临床试验时,识别端点极有帮助。
而本文的重点是关系到适当的冻结和准备组织的各种研究,我们的合作小组还讨论了有用的端点的肌肉标本的病理分析。目前,正在执行的病理分析时采用的方法没有官方的共识,以及各种不同的研究分析,以与新的发现之前的出版物为每个相应的疾病。因此,我们认为这将是有益的建议在肌肉病理特征病理端点的规划一些一般性的准则。前量化的一项研究的病理学,相当大的思想应放入1)纤维尺寸测量的方法,2)的纤维类型特定异常或治疗效果的可能性,3)异常或影响的可能性被限制个别的肌肉,和4)的策略用于定量病理结果是特征的疾病正在研究中。纤维的大小是一个必要的端点大多数研究,遗憾的是在它是如何量化广泛的变化。许多研究利用专有的图像处理软件提供的自动定量方法报告这些结果,但许多这些项目的拍摄快捷键(如假设纤维是圆形或椭圆形),可以使这些自动测量不准确。有必要了解这些自动化的程序是如何让他们的测量结果b安伏有信心的测量,我们鼓励研究者,包括纸张的方法这些细节。此外,用于表示纤维大小的特定的测量是非常可变的,有些测量是优选他人10,11。纤维的大小的常用测量纤维横截面面积(CSA),尤其是因为进行生理学研究调查其结果标准化,以使用其工具获得CSA测量。不幸的是,虽然CSA测量可以准确地反映纤维尺寸完美横截面,它们广泛地依赖于纤维方向(的范围内,纵向或斜向剖面的纤维将有虚高CSA测量)和对纤维的大小从而不理想的测量。纤维尺寸的优选测量其较少依赖于纤维的截面积为最小的Feret直径(MinFeret直径),这是个测量e小调直径在肌肉细胞12。这种测量方法是仅稍微依赖于纤维的取向和一般临床黄金标准纤维的测量,调查被鼓励移向使用这种技术。这些测量通常可以使用其生成CSA测量值13中的相同的软件制造,并且也简单的手动测量。对于根据光纤类型,特定的肌肉,并在涉及到具体的疾病病理结果的情况下评估的病理资料,这些都是应该只是一个研究的规划过程中考虑较少争议的问题。纤维类型可以用免疫组织化学或ATP酶染色法进行评价,但要考虑到特定的肌肉和动物物种具有这些纤维类型的特定混合物(因而需要不同的期望和测试策略)是有用的。肌肉特异性病理介入或治疗功效可以发生,并且总肌重量与对照相比,可以用来决定肌肉病理评估之前,以确定疾病的异质性程度。最后,这是众所周知的,许多肌肉 疾病都与特定的病理异常(如在杆状体肌病杆状体杆)14,15相关联,因此它也是有用的,考虑这些异常是否被发现在纤维型或肌在进行分析时,16,17的具体分布。总体而言,虽然我们不建议一个不灵活的一套肌肉的评价标准,但我们相信这些问题之前,应在任何骨骼肌肉疾病的病理研究的性能考虑。
The authors have nothing to disclose.
This publication is funded through Cure CMD, an Association Contre Les Myopathies (AFM) grant (project 16297), and the National Institutes of Health (grant numbers K08 AR059750 and L40 AR057721). We would also like to thank Dr. Julie Tetzlaff for her assistance in revising this manuscript.
For tissue freezing | |||
Liquid nitrogen Dewar with a liquid withdrawal device | Custom Biogenic Systems | Lab-5 Series | Any other liquid nitrogen dewar could substitute. |
Cold-conductive container | Fisher | 02-583A | |
Wide insulated container capable of holding liquid nitrogen | Nalgene | 4150-2000 | |
Oakton Temp 10 Thermocouple Thermometer | Thomas Scientific | 1228Y01 | Optional, but can be very useful in identifying the point at which isopentane is freezing. |
For single fiber functional testing | |||
Petri dish | Fisher Scientific | 08-757-11 | |
Sylgard coating | Ellsworth Adhesives | 3-6636 | |
Dissection stereomicroscope | Harvard Apparatus | 693101 | Any other dissection microscope could substitute. |
For cell culture | |||
Sterile laminar flow hood | Thermo Scientific | 51022485 | Any other cell culture hood could substitute. |
Materials | |||
For tissue freezing | |||
Plastic bags/containers | Nasco | B01009WA | |
Thermal safety gloves | Denville | G4162 | |
Face shield | Fisher Scientific | S47640 | |
Long forceps | Fisher Scientific | 12-460-807 | |
Marker | Staples | 125328 | |
For cell culture | |||
Sterile 50 mL conical tubes | Denville | C1060-P | |
PES filter unit, 500 mL, 0.22 µm | Denville | F5227 | |
Sterile forceps | Fisher Scientific | 644320 | |
Ice packs | Fisher Scientific | NC9909223 | |
Insulated Styrofoam box | Polar Tech | 207F | |
Packing tape | Staples | 795570 | |
Reagents | |||
Tissue Freezing | |||
Liquid nitrogen | Airgas | NI NF160LT350 | Store liquid nitrogen in containers designed for low-temperature liquids |
Isopentane (2-methylbutane) | Sigma | M32631-4L | Store Isopentane in a flammable materials cabinet. |
EM fixative (choose one) | The buffers without fixative should also be available, as EM-fixed tissues should be transferred from fixative to buffer after several hours or days if they are not immediately processed for EM. | ||
2.5% glutaraldehyde in 0.1M cacodylate buffer | Electron Microscopy Sciences | 16537-15 | |
Karnovsky's fixative (3% glutaraldehyde + 2% paraformaldehyde in 0.1M phosphate buffer) | Electron Microscopy Sciences | 15732-10 | |
For functional studies (if desired) | |||
Relaxing solution, containing (in mmol/L) | |||
40 BES | Sigma | B9879 | |
10 EGTA | Sigma | E4378 | |
6.56 MgCl2 | Sigma | M8266 | |
5.88 Na-ATP | Sigma | A3377 | |
1.0 DTT | RPI | D11000 | |
46.35 K-propionate | Make a solution by mixing proprionic acid (Sigma P1386) and KOH (Fisher P250) 1:1 | ||
15 creatine phosphate, pH 7.0 | Sigma | P7936 | |
0.4 leupeptin | Calbiochem | 10895 | |
0.1 PMSF | Sigma | P7676 | |
Skinning solution, containing | |||
Relaxing solution (See Above) | |||
0.5% Triton X-100 | Sigma | T8787 | |
Indicating silica granules | |||
Flat pieces of cork (1 x 1 inch) | Electron Microscopy Sciences | 63305 | |
Glycerol | Sigma | G2025 | |
For cell culture (if desired) | |||
Complete Growth Medium, containing (for 500 mL) | Sterilize by filtering the solution through a 500 mL 0.22 µm PES filter unit. Store at 4°C and use within 1 month. | ||
395 mL of high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) | Sigma | D5671 | |
100 mL of fetal bovine serum (FBS) | Sigma | F6178 | |
5 mL of 100X Penicillin-Streptomycin-Glutamine (PSG) | Sigma | G1146 | |
Storage requriements | |||
See the materials safety data sheet (MSDS) for all other reagents for storage specifications |