この研究の彼らのサポートのベッチャー財団と筋萎縮性側索硬化症協会 (#17-II-344) に感謝しております。

マウスを含むすべてのプロシージャは、コロラド大学アンシュッツ医療キャンパス機関動物ケアおよび使用委員会によって承認された (91813(05)1D)。
 1 です一日 1: プライマリ (1 °) ウシ血清アルブミン (BSA) ブロック染色 <ol><li>マウスの後肢筋 (例えば、前脛骨筋、ヒラメ筋) の空気乾燥凍結切片の荷電のスライドに取付けた ~ 30 分。 11. 疎水性障壁 PAP のペンを使用してセクションに境界線を描画します</li>。
	<li>場所 ~ 5 %bsa/リン酸緩衝生理食塩水 (BSA/PBS) 非特異抗体の結合をブロックする各スライド上を 250 μ l 添加します。スライド 1 のため室温で孵化させなさい</li>
	<li>ブロック中準備 1:50 5% 培養上清 1 ° 抗体の希釈 BSA/PBS (すべてはマウス モノクローナル抗体; 表 1 6 , 12 を参照してください); の 250 μ L を準備スライドごとのソリューションです。氷に 1 ° 抗体株式および希薄を維持</li>
</ol> <img alt="Table 1"src="/files/ftp_upload/56024/56024table1.jpg"/> テーブル 1: 一次抗体 MyHCs を区別するために使用します。
<ol start="4"> <li>5 %bsa/PBS ブロッキング液の吸引は、次の適切なスライドに 1 ° 抗体希釈の 250 μ L を追加。追加 250 μ L の 5 %bsa/PBS (2 °) 二次抗体だけネガティブ コントロールをスライドです</li>。
	4 ° C、湿度の高い環境で 24-48 h で <li>加温します 。
	注: 湿らせたろ紙をアルミ箔で覆われてペトリ皿は適切なインキュベーション室として役立つかもしれないです</li>。
</ol> 2。日 2:2 ° 蛍光抗体染色 <ol><li>1 ° 抗体溶液を吸引または 5 %bsa/PBS 溶液を制御します。洗ってすべてスライド 3 回、10 分の 5 %bsa</li>。
	<li>洗濯中準備 5% の精製、fluorophore 共役 2 ° 抗体の希薄を 1: 200 BSA/PBS (表 2 を参照してくださいすべてがヤギ抗マウス)。スライドごとに解決策の 250 μ L を準備します。氷で暗闇の中 2 ° 蛍光標識抗体を保持</li>
</ol> <img alt="Table 2"src="/files/ftp_upload/56024/56024table2.jpg"/> テーブル 2: 蛍光標識二次抗体を視覚化する一次抗体認識の MyHCs するために使用します。
<ol start="3"> <li>5 %bsa/PBS ブロック ソリューションの 3 番目のアプリケーションの吸引は、次のすべてのスライドに 2 ° 抗体希釈の 250 μ L を追加。暗く、湿気の多い環境で室温で 90 分間インキュベートします</li>。
	<li>吸引 2 ° 抗体溶液。洗ってすべてスライド 3 回 10 分 5% の BSA/PBS</li>。
	PBS の <li>リンス。10 分間乾燥</li>
	<li>マウント非常任理事、低粘度の水性取付中 coverglass</li>。
	<li>、乾燥時は、マニキュアとスライド エッジをシールします。乾燥し、暗い slidebox に格納します</li>。
</ol> 3。落射蛍光顕微鏡でスライドをイメージング <ol><li>きれいに拭きます小、70% エタノールに浸したラボ - スライドです</li>。
	<li>写真装置 (材料の表 を参照) を搭載した落射蛍光顕微鏡を使って immunostained 筋セクションのデジタル画像を取得します。
	<ol><li>約 1 mm 2 (10 × 対物、1.4 NA) の領域を選択します</li>。
		<li>ビューを介して、505 アレクサ 488 共役 2 ° 抗体蛍光 nm ロングパス ・ フィルター。595 nm ロングパス ・ フィルターを介して Alexa 594 共役 2 ° 抗体の励起による蛍光を表示します。互換性のあるイメージング ソフトウェアを搭載した PC のコンピューターで画像をデジタル化します。イメージング ソフトウェアを後で分析からスケール バーが含まれます</li>。
	</ol></li></ol> 4。フィジー画像の解析 注: 解析に進む、前にフィジー (健康の国民の協会、ベセスダ、MD から自由に利用可能) は、https://imagej.net/Fiji/Downloads 経由でインストールする必要があります。また、このプロセスで使用するマクロは、補足のファイル で提供されます。マクロ ファイルを簡単にアクセスできるディレクトリに配置します。
フィジーで選択したセクションの <ol><li>ロード、明視野イメージ。プラグインに移動 &#8594; 分割 &#8594; Trainable ウェカ ・ セグメンテーション</li>。
	、光ファイバーの線を描画し、セクションの細胞領域を規定するために <li>&#34; クラス 1 に追加 &#34;。繊維間空間に線を描画し、その後、&#34; クラス 2 に追加 &#34; 細胞外ドメインをマークします。各クラスで 5-10 ラベルがあるまでを繰り返します。
	<ol><li>クリック鉄道分類子。結果の赤と緑のオーバーレイで手動で複数のラベルを追加 (手順 4.2 参照)、必要な場合は、イメージの正しく分割部分に。繊維 (赤) は、繊維 (グリーン) 間のスペースから適切に分離されてまで繰り返します</li>。
	</ol></li> <li>クリックして &#34; 取得確率 &#34; と新しくラベル付けされたフォルダーに画像を保存します</li>。
	<li>は、同じフィールドから撮影した蛍光画像と (手順 4.2 4.3) の上記の手順を繰り返します。ラベルの immunostained 繊維として &#34; クラス 1 &#34; とすべての非蛍光性領域として &#34; クラス 2 &#34;。結果の確率マップを加工し、明視野イメージが格納されている同じフォルダーに保存します</li>。
	<li>はフィジーで以前に保存した確率マップの 1 つを開きます。イメージング ソフトウェアによって提供されるスケール バーの直線を描画します。分析に行く &#8594; スケールを設定します。スケール バーで指定された適切な値 (すなわち、既知の距離、長さの単位) パラメーターを変更し、チェック、&#34; グローバル &#34; 各イメージの尺度を標準化します</li>。
	<li>のプラグインに移動 &#8594; マクロ &#8594; を実行 &#8594; Tyagi ら 繊維定量 macro.ijm (補足ファイル を参照してください)。すぐに、ナビゲーション ウィンドウが表示されます。画像を開く &#39; s ホスト フォルダー;ダイアログ ボックスが表示されます。各ダイアログ ボックスの変更、&#34; 背景 &#34; ドロップ ダウン値 &#34; 光 &#34; 注: フォルダーが繊維アウトラインの画像と (CSA ・ フェレー径に最も関連する結果のスプレッドシート ソフトウェア ファイルが表示されます繊維形態の定量化測定されたパラメーターで調整できます設定測定の分析)。フィジーの細孔解析関数によって生成された繊維形態データをスプレッドシート ソフトウェア両方の蛍光および明るいフィールド画像のファイルに自動的に転送されます</li>。
	<li>蛍光繊維分野で対応する明視野イメージで総繊維数の数を割ることによってフィールドで指定された MyHC を表現する繊維の割合を計算します</li>。
</ol>

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著者は開示する利害の対立があります。

ここでは、骨格筋線維の種類を識別するための有用な方向を提供しております。そうすることで、データの解析のための新たなアルゴリズムについて述べる.
我々 の結果は主前レポート5,8,10のものを確認して、私たち自身の手動測定を反映、アルゴリズムが正確に表示されます。まだ、アーティファ?...

それは、骨格筋は多くのタイプ1の単繊維から成るいくつかの時間のために知られています。当初は、繊維の 2 つのグループがその収縮特性に基づく特徴付けられると名前は、適切に、遅筋 (タイプ I) と速筋 (タイプ II)。これらのカテゴリは、繊維の代謝に基づくさらに区別されていました。以来タイプ I 繊維はミトコンドリアで豊富であり、酸化的代謝に依存して、彼らを力強く肯定的なニコチン酸アミドアデニンジヌクレオチド ニコチンアミドアデニンジヌクレオチドテトラゾリウム還元酵素 (NADH-TR) のジアホラーゼ2またはコハク酸脱水素酵素 (SDH) の3で解明しました。染色。対照的に、タイプ II 線維展示低い NADH TR ジアホラーゼの SDH 可変度染色と速筋の 2 つのサブグループに分けられた (タイプ IIA、IIB 型) の相対的な酸化能力に基づいてややぞんざい。繊維のこれらの違いは、ミオシン ATPase 染色によるより効果的に可視化されている型、pH 4.0 で前培養後繊維染色暗いとタイプ IIB 線維はタイプ IIA 線維で pH 10.0 に沈殿物次中古インキュベーションを吸収します。4を染色中間。
最近では、ミオシン重鎖 (MyHC) 成分の免疫組織化学的染色繊維タイプ5の細かい識別子として浮上しています。タイプ I、IIA 型し、タイプ IIB 線維がすべて識別する精度の MyHC プロファイルに基づきます。さらに、別の速筋代謝中間体繊維の種類は識別された6されている IIX を入力します。1 つ以上の MyHC を表現するハイブリッド繊維また確認された5,7,8をされています。猫やヒヒなどいくつかの種は、エクスプレス タイプ IIB MyHCs6にない知られています。MyHC 免疫染色は筋組成の最新の評価では現在、この手法で得られたデータの解析は面倒で時間のかかる自動支援なし。このため、半自動化された方法でこれらのデータを分析する一握りは先進5,9,10をされています。ここでは、半自動化された小説と一緒に筋線維タイプ5,7,8,10,の免疫組織化学的同定のため比較的標準的なプロトコルを提案します。アルゴリズムの精度で光ファイバー型および繊維の形態の分析を加速します。

<table><tbody><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418-100G</td><td>5% in PBS</td></tr><tr><td>Hydrophobic Barrier Pap Pen</td><td>Scientific Device Laboratory</td><td>9804-02</td><td></td></tr><tr><td>Microscope Slides</td><td>Globe Scientific</td><td>1358W</td><td></td></tr><tr><td>Coverglass</td><td>Fisher Scientific</td><td>12-544-E</td><td></td></tr><tr><td>Immumount</td><td>Thermo Scientific</td><td>9990402</td><td></td></tr><tr><td>Nail Polish</td><td>L&#039;Oreal</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td><td></td><td>Discontinued</td><td></td></tr><tr><td>SPOT RT/KE</td><td>SPOT Imaging Solutions</td><td>RT940</td><td></td></tr><tr><td>Dell Optiplex</td><td></td><td></td><td></td></tr><tr><td>BA-F8 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgG2b; 1:50</td></tr><tr><td>SC-71 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monclonal mouse IgG1; 1:50</td></tr><tr><td>BF-F3 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgGM; 1:50</td></tr><tr><td>6H1 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgGM; 1:50</td></tr><tr><td>Alexa Fluor 594 anti-IgG2b</td><td>Invitrogen</td><td>A21145</td><td>goat anti-mouse; 1:200</td></tr><tr><td>Alexa Fluor 488 anti-IgG1</td><td>Invitrogen</td><td>A21121</td><td>goat anti-mouse;1:200</td></tr><tr><td>Alexa Fluor 594 anti-IgGM</td><td>Invitrogen</td><td>A21044</td><td>goat anti-mouse;1:200</td></tr><tr><td>OCT</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>isopentane</td><td>Fisher Scientific</td><td>O3551-4</td><td>cool with liguid nitrogen</td></tr><tr><td>PBS</td><td>Fisher Bioreagents</td><td>BP665-1</td><td>10x, dilute to 1x</td></tr><tr><td>Kim wipes</td><td>Kimberly-Clark</td><td>06-666A</td><td></td></tr></tbody></table>

semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition

長年、骨格筋線維のタイプ間の区別されたミオシン ATPase 染色による視覚化に最適。最近では、ミオシン重鎖 (MyHC) 成分の免疫組織化学的染色繊維型の細かい識別子として浮上しています。タイプ I、IIA、IIX を入力を入力し、タイプ IIB 線維は、MyHC プロファイルに基づく精度で今識別できます。ただし、これらのデータの手動解析が遅いと右下をすることができます退屈。この点では、繊維の種類組成と形態の迅速で正確な評価は非常に望ましいツールです。ここでは、最新の免疫組織化学染色繊維の種類や繊維の形態の分析を加速する新たな半自動アルゴリズムとのコンサートでマウスの後肢筋から得られた凍結切片における MyHCs のためのプロトコルを提案する.ヒラメ筋が表示されます染色タイプ I およびタイプ IIA 線維、予想通り、ではなく、IIX またはタイプ IIB 線維。その一方で、前脛骨筋 IIX とタイプ IIB 線維、タイプ IIA 線維のごく一部の主にで構成されていた、またはほとんどないタイプ I 線維。いくつかのイメージの変換は、繊維形態を (すなわち、断面積 (CSA)、極大値と極小のフェレー径) のさまざまな側面を測定する目的について確率マップを生成する使用されました。これらのパラメーターが手動で得られた値と比較し、得られた値。CSA、最大または最小のフェレー径に関して解析のいずれかのモード間に有意差は認められなかった (すべてp &#62; 0.05)、提案手法の精度を示します。したがって、私たちの免疫染色解析プロトコルは高齢化とミオパチーの多くのモデルで筋肉の組成に及ぼす影響の調査に適用できます。

後肢筋 (すなわち、前脛骨筋、ヒラメ筋) 年齢不詳の男性 c57bl/6 マウスから解剖されたフラッシュ 10 月に筋肉を含むプラスチック金型の沈没によって固定されている液体窒素冷却イソペンタン化合物。8-10 μ m の切片が、cryotome を使用して、-20 ° C でカットし、異なる荷電ガラス スライド12に転送します。
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Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

骨格筋線維タイプの新式の識別子として浮上している免疫組織化学的染色ミオシン重鎖アイソ フォームの (すなわち、タイプ I、IIA を入力、IIX を入力、IIB を入力)。ここでは、繊維の種類や繊維の形態の迅速な評価を容易にする新しい半自動アルゴリズムとともに汚損のプロトコルを提案する.

マウス骨格筋形態と繊維の種類組成の半自動解析

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

semi-automated-analysis-mouse-skeletal-muscle-morphology-fiber-type

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.4K ビュー.  University of Colorado School of Medicine. 骨格筋線維タイプの新式の識別子として浮上している免疫組織化学的染色ミオシン重鎖アイソ フォームの (すなわち、タイプ I、IIA を入力、IIX を入力、IIB を入力)。ここでは、繊維の種類や繊維の形態の迅速な評価を容易にする新しい半自動アルゴリズムとともに汚損のプロトコルを提案する.

ビデオ: Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

単離骨格筋のミオ - 機械的分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

医学

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

プロテオミクスから骨格筋の性別二型

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

経胸壁心エコー検査による肺動脈狭窄のマウスモデルにおける右室の構造と機能の評価

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

MyDoBID- 筋線維の種類を効率的に特定するための新しい技術

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

著者スポットライト:MyDoBID技術を使用した骨格筋線維タイプの謎の解読 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

ヒト骨格筋の線維型同定

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

生化学

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

全山神経筋接合部の免疫蛍光解析と形態測定解析のための単一骨格筋線維の解剖

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

神経科学

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.

骨格筋中の脂肪滴含量の線維タイプおよび細胞下特異的分析

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

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

ミオシン重鎖免疫組織化学を使用して、ラット筋断面における筋線維人口分析のための迅速な自動化されたプロトコル

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

生物学

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

光学系を用いたヒドロゲル包埋無傷マウス筋線維のハイスループット収縮測定(英語)

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

マウス骨格筋形態と繊維の種類組成の半自動解析

要約

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単離骨格筋のミオ - 機械的分析

プロテオミクスから骨格筋の性別二型

経胸壁心エコー検査による肺動脈狭窄のマウスモデルにおける右室の構造と機能の評価

ヒト骨格筋の線維型同定

ヒト骨格筋の線維型同定

ヒト骨格筋の線維型同定

全山神経筋接合部の免疫蛍光解析と形態測定解析のための単一骨格筋線維の解剖

骨格筋中の脂肪滴含量の線維タイプおよび細胞下特異的分析

ミオシン重鎖免疫組織化学を使用して、ラット筋断面における筋線維人口分析のための迅速な自動化されたプロトコル

光学系を用いたヒドロゲル包埋無傷マウス筋線維のハイスループット収縮測定(英語)