We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1。组织收获和肌肉组织的冷冻<ol><li>安乐死的小鼠用异氟烷（2-氯-2-（二氟甲氧基）-1,1,1-三氟乙烷）的过量。执行此步骤在通风橱中并利用一第二容器的方法，如干燥器或钟罩含有棉签浸泡在异氟醚。 </li><li>用力挤压脚垫确认死亡。使用安乐死的辅助方法，如颈椎脱位。 </li><li>湿用70％酒精的皮毛，以减少对肌肉松弛头发缕粘着。用细镊子，如＃5剥离皮肤脱落的肢体。 </li><li>小心地从骨骼中分离的兴趣（）在这种情况下，胫骨前（TA）的肌肉的下面，从肌腱开始并移除。 </li><li>切除的肌肉，它覆盖在华侨城（最佳切削温度化合物），并把它平放在一个标有一次性冻结模具。确保肌肉在其正常的生理方向（头至大l）在不拉伸。可替代地，冻结小发泡胶正方形肌肉和利用昆虫针以确保肌肉的正确长度针它。 </li><li>寒意2-甲基丁烷（异戊烷）中采用液氮钢制烧杯中。这平均需要3 - 5分钟。不断搅拌间歇直至白色沉淀开始形成在烧杯的底部和壁上。 注意:当这种情况发生时，异戊烷已达到最佳冷冻温度（-150℃） </li><li>浸含在冷冻2-甲基丁烷肌肉的模具。冷冻小肌肉，如隔膜和趾长伸肌（EDL）6 - 12秒，较大的肌肉如腓肠肌比目鱼（GS）和肱三头肌15 - 20秒。不要冷冻肌肉太久，因为这可能会导致开裂。 </li><li>转让的冷冻组织干冰，让异戊烷蒸发（注意，此过程平均需要大约15 - 20分钟）。包裹组织中铝箔并将其存储在üLTRA低温（-70℃〜-80℃），直到切片。 </li></ol> 2.嵌入组织在OCT准备组织块<ol><li>干冰运输组织，以cyrostat防止解冻。转移组织到低温恒温室（设定为-20至-24℃，以避免组织的充分解冻），并让他们平衡30分钟。 </li><li>打开珀耳帖冷却海湾上。如果低温恒温器不具备这种冷却，使用喷雾冷却喷雾快速冻结十月不要让肌肉解冻的任何一点的嵌入，因为这可能会导致冻害期间。 </li><li>添加均匀的薄层华侨城在试样上盘，让它冷却。当大多数的OCT被冻结在底部，同时仍然液体在顶部，插入在正确的方向的组织（垂直华侨城为横截面和平行华侨城为纵向部分）。使用喷雾冷却喷雾迅速冷冻十月触摸组织unembeded部分热转ractor并等待45秒。 </li><li>添加另一个薄层OCT的周围的肌肉，喷洒气溶胶冷却喷雾和提取的热量作为在上一步。 </li><li>在保持小幅度增加OCT和快速使用气溶胶喷雾冷却和加热萃取的结合冻结华侨城，直到整个肌肉被覆盖。 </li><li>放的热量提取的组织块的顶部，等待5分钟。 </li></ol> 3.准备部分和肌肉半山区肚识别章节<ol><li>安装试样到样品头。拧紧旋钮固定盘。确保在试样圆盘与所述样品头良好的接触。 </li><li>调节室内温度°之间-21和-24℃，等到设定温度达到。 </li><li>其厚度调节设置（7微米）;修剪通过推进或使用粗，细设置缩回块的块。 </li><li>收集部分通过按滑动的顶部上的部分中，使用相反电荷之间的吸引力，以方便切割部分以滑动的粘附预热带正电的显微镜载玻片。 </li><li>通过测定各部分的横截面面积（CSA）识别中期腹部分。连接到显微镜的计算机上执行此操作使用图像处理软件。 注:相衬图像的拍摄和整个肌肉的圆周经由上的软件程序跟踪功能测定。这将产生两个CSA以及最小鼬直径（测量纤维横截面尺寸的被表示为对一个粒子相反两侧的两个平行切线之间的最小距离）的测量。注意:当CSA和鼬最小直径测量开始高原，肚皮中旬已经达到。 </li><li>库存和归档的幻灯片在-80℃的未来染色。 注意:OCT不需要之前被除去以stainin摹程序​​。一旦载玻片盖上盖玻片，他们准备立即染色。 </li></ol> 4.染色注:虽然详细的一步一步的程序，可以在产品数据表中找到，并应遵循染色程序，个人优化可能需要以获得最佳的染色。 <ol><li>因为安装介质（见表）是不与水混溶的，通过分级醇增加脱水载玻片（每次2分钟，70％，95％和100％的乙醇），并清除它们用二甲苯（100％，5分钟），以确保幻灯片不会成为不透明随着时间的推移。 </li><li>盖玻片部分，并让他们干。图片显微镜配备图像处理软件。 </li></ol>

<ol>
	<li>Bonnemann, C. G., 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=Diagnostic+approach+to+the+congenital+muscular+dystrophies.">Diagnostic approach to the congenital muscular dystrophies.</a> Neuromuscular disorders : NMD. 24, 289-311 (2014).</li><li>Caughey, J. E., Pachomov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diaphragm+in+dystrophia+myotonica.">The diaphragm in dystrophia myotonica.</a> J Neurol Neurosurg Psychiatry. 22, 311-313 (1959).</li><li>Fetterman, G. H., Wratney, M. J., Donaldson, J. S., Danowski, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophy.+I.+History,+clinical+status,+muscle+strength,+and+biopsy+findings.">Muscular dystrophy. I. History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li><li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy.">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li><li>Mercuri, E., 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=Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine.">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li><li>Tasca, G., 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=Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles.">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779 (2012).</li><li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, T., 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=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. 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The authors have nothing to disclose.

组织学和免疫组化已被用作主要的工具在理解表现和病理的进展在各种神经肌肉疾病。经典地，肌肉活检第一固定在甲醛，然后用石蜡包埋蜡。虽然甲醛固定保持组织结构，并允许组织存储和运输，在RT，这也灭活酶和蛋白质交联迫使进一步措施，以实现精确的免疫染色。这个问题与使用冰冻切片消除，但这种方法提出了获得最佳的染色效果和准确的诠释14,15独特的技术挑战。 <p class="j...

检查肌肉组织学中起着不同类型神经肌肉疾病1-4中的认识至关重要的作用。当与其他技术相结合，它使研究人员能够更好地了解病理的表现和进展的，也可以是必需评估治疗干预5-10的功效。然而，要准确，组织需要以适当的方式被保留以便切除之前立即保持生理状态。这种去除，保存，切片，和染色过程中需要非常小心。 组织可以以两种不同的方式进行光学显微镜研究来制备。第一种方法，福尔马林固定的石蜡包埋（FFPE）的部分，需要组织到被嵌入石蜡11,12之前被固定在10％的天然缓冲甲醛。固定步骤，有助于更好地保存形态，而且交联内源性PRoteins因而需要复杂的抗原修复程序用于免疫染色和消除酶染色对这些部分12的可能性。冰冻切片，另一方面，不需要进行预固定或处理;因此，蛋白质能保留原生生物构13,14。此外，冷冻切片也允许快速的周转时间，这有时是必要的，例如在肉瘤和其他外科活组织检查15的术中诊断。但是，如果没有正确完成，该方法是容易冻结损坏，它引入了改变肌肉结构使得部分不适宜用于组织学检查。本文将概述准备肌肉活检，以达到正确检查最佳染色最有效的方法。

<table><tbody><tr><td>Cryostat</td><td>Leica CM 1850</td><td>Leica Microsystems Inc.</td><td>Buffalo Grove, IL 60089 U.S.A., office phone 1-800-248-0123&lt;br /&gt;Alternative: Richard Allen Scientific</td></tr><tr><td>Microscope</td><td>Nikon Eclipse 50i</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064 U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternative: Olympus</td></tr><tr><td>Image analysis software</td><td>Nikon Imaging Software Basic Research</td><td>Nikon Instruments Inc.</td><td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A., Phone: 1-631-547-8500&lt;br /&gt;Alternatives: Image J, Metamorph</td></tr><tr><td>Isoflurane</td><td>14043-220-05</td><td>JD Medical Dist. Co., Inc.</td><td>1923 West Peoria Avenue, Phoenix, AZ 85029 U.S.A.</td></tr><tr><td>OCT</td><td>4583</td><td>Sakura Inc.</td><td>Torrence, CA 90501 U.S.A.&lt;br /&gt;Alternative source: Leica Microsystem</td></tr><tr><td>Freeze It</td><td>23-022524</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA, 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Disposable tissue mold</td><td>22-363-554</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Colorfrost plus slides</td><td>9991001</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Acetone</td><td>650501-4L</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Ethyl alcohol</td><td>HC-1300-1GL</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>2-methyl butane</td><td>M 32631-SL</td><td>Sigma-Aldrich</td><td>3050 Spruce Street, St. Louis, MO 63103 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Xylene</td><td>HC-7001GA</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Cytoseal 280</td><td>8311-4</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Hematoxylin Gill #3</td><td>CS402-1D</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr><tr><td>Eosin</td><td>6766007</td><td>Thermofisher</td><td>790 Memorial Dr, Cambridge, MA 02139 U.S.A.&lt;br /&gt;Alternative source: VWR Scientific</td></tr></tbody></table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

肌肉活检组织学评价曾担任在神经肌肉疾病的病理发展过程的理解不可或缺的工具。然而，为了这样做，适当的照料需要切除并保存组织，以达到最佳的染色时，必须考虑。组织保存的一种方法包括固定在甲醛组织，然后用石蜡包埋它们。此方法保留形态良好，并且允许长期储存于RT但是麻烦的，并且需要处理的有毒化学品。此外，甲醛固定导致抗原的交联，这需要抗原修复协议有效免疫染色。与此相反，冷冻切片不需要固定，如此保留生物抗原的构象。此方法还提供了一个明显的优势在周围时间快转，使得它在情况需要像intraopera快速组织学评估特别有用略去手术活检。这里，我们描述制备肌肉活检用于可视化具有不同的组织学和免疫染色的最有效的方法。

该组向上收获和组织冷冻可以看出，在图1中 。切下的组织应存储在浸泡在PBS中，以避免在干燥（ 图1A）纱布。当组织被选择以用于组织学分析，他们应该被浸入OCT和布置在低温模具的正确方向，并尽可能接近生理长度尽可能以确保准确的形态（ 图1B）。异戊烷适于组织冷冻的浆液可以通过冷却的异戊烷在液氮中，并搅拌，直到小白色沉淀出现在底部来实现?...

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

do-s-don-ts-preparation-muscle-cryosections-for-histological

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JoVE Journal

Medicine

31.7K 次观看.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

视频: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

闪存冻结和Cryosectioning E12.5小鼠脑

科研

生物学

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 ...

发育生物学

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

用于神经肌肉分析的大鼠声带的制备

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

神经科学

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

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.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

骨骼肌中脂质液滴含量的纤维类型和亚细胞特异性分析

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

医学

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

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

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

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

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

肌肉干细胞中自噬的原位免疫荧光染色

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.