研究は、シカゴの糖尿病研究研修センター（動物モデルコア）、およびKovlerファミリー財団からの贈り物の大学に米国公衆衛生サービスグラントDK - 081527、DK - 072473とDK - 20595によってサポートされています。

第1部：試験片の調製<ol><li>解剖顕微鏡下でマウスを置く、まだ接続されている十二指腸と脾臓と一緒に全体の膵臓を削除して、事前に計量したガラススライド上に臓器を置きます。それは膵臓にバインドされている側に沿って結合組織を切断して十二指腸を削除します。膵臓が完全にスライドで隔離されるまで、膵臓で脾臓や余分な脂肪をカットして取り外します。 （膵組織の少し暗いとタナー色から分化したと膵脂肪は、その白い色で区別できる。） </li><li>それが完全に覆われるように膵臓上、カバースリップを（標準のカバーガラス25x75mm 50x75mm大型カバーガラス）に置きます。スライドとカバーの間に膵臓を平坦にする（重い本など）の重量を使用してください。重量に応じて、膵臓を平坦化すること約5分かかります。 </li><li>重量をはずしなさい。膵臓は、スライドに対しても平坦化の場合は、4℃で一晩4％パラホルムアルデヒド（PFA）に置きます。午前中は、カバーガラスを取り除き、よく日中のPFAに膵臓全体を（6-8時間）に公開。膵臓が十分に固定されていない場合、それは、長いPFAでおそらく一晩おくことができます。一晩（PBS）リン酸緩衝生理食塩水にトリトンX - 100 1％でコンテナにスライドを移し。翌日、1〜2日のために飽和ショ糖の容器にスライドを転送する。その後、蛍光シグナルの最適な解像度を可能にする組織がクリアされるまで100％のグリセロールの容器、にスライドを移転すること、組織の清算は、約1-3日かかります。 （蛍光シグナルは、数週間、および月間持続することがありますが、組織が最適な解決のためにクリアされた後の画像をすぐに実行する必要があります。） </li><li>オルガンとその蛍光シグナルを維持するために暗く、涼しい場所で膵臓を保管してください。 </li></ol>第2部：画像及び無傷の膵臓のβ細胞の面積を定量化<ol><li>ピンセットや手袋をはめた手を使って、100％のグリセロールとコンテナのスライドを取り出します。過剰なグリセロールを拭き取り、排他的にエタノールでカバーガラスの背面をクリーニングによりイメージングのためのスライドを準備します。 </li><li> StereoInvestigatorソフトウェア（MicroBrightField、ウィリストン、バーモント州）を開き、3つの大きなガラスのスライド上で数週間、または2つの25ミリメートルカバーガラスの間に押された3週間より膵臓若いイメージングマウスの10倍の対物レンズより膵臓古いイメージングマウスの2倍の対物レンズを選択します。画像（イメージ→ライブイメージを）生きるためにそれを設定して、イメージを視覚化する。明らかにGFP -タグ付きβ-細胞を示して露光時間を選択してください。 （画像をover​​exposingには、追加の偽信号を作成しながら画像をUnderexposingと、小さな島とβ-細胞を除きます。） </li><li>基準点を確立するために画面上の任意の場所をクリックし、全体の膵臓の周りに輪郭を描画するために進んでください。仮想スライスのプロセス（画像→仮想スライスを取得）を開始します。最高の焦点面、すなわち、フォーカスノブをスクロールしてZ軸上で可視的を絞った島とβ-細胞の数が最も多い平面を決定し、選択します。選択後は、StereoInvestigatorは自動的に画面に表示された画像をキャプチャします。電動ステージで、仮想スライス機能は、連続して描かれた輪郭（図1A）内に個別のイメージとしてそれぞれの光学パネルをキャプチャして、1つのマージされた画像（図1B）として、それらのすべてを兼ね備えています。仮想スライスイメージングは​​、落射蛍光コンフィギュレーションを使用して、最終的なバーチャルスライスが最大ではない統合された二次元画像になるようにカメラがない完璧にピントのものも含めて一定の深さに存在するすべての信号を、キャプチャすることができますすなわち、広視野フィルタ膵臓全体の三次元再構成からの投影。平均的な仮想スライスのスキャン時間は、サンプルあたり30分ですが、大規模な膵臓のために時間かかることがあります。 </li><li>画像を保存した後、ImageJを使用してイメージを開放することで、その分析を開始。画像のロードが完了し、画面に表示されたら、仮想スライス分析マクロを（開いて実行<a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">補足資料1</a> ）。 </li><li>マクロで提供されるステップバイステップの指示に従ってください。 &quot;魔法の杖&quot;と&quot;範囲選択&quot;ツールを使用して光散乱によって作成された不要なアーチファクトを除去する。画像から不要な背景を確認し、引く。その後のローリングボールの機能が効果的に自家蛍光や散乱光を除去できるように、&quot;ライン&quot;ツールとの最大の膵島の半径を決定する。 β-細胞の小さな島とクラスターの蛍光シグナルを捕捉するために最も適切な&quot;低しきい値&quot;をテストし、選択（&lt;3 × 10 4μmの2）。膵島の蛍光シグナルを捕捉するために最も適切な&quot;最大しきい値&quot;をテストし、選択してください。測定される仮想スライス、の定量化を実行します。と膵島面積、周囲、真円度、およびフェレーの直径のリストを生成します。 </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="図1a" /> そのまま成人の膵臓のβ細胞の全体的な分布の図1A仮想スライスの画像解析 10週の雄マウスの膵臓の個々の光学パネルの画像は、2倍の目標の下で撮影。イメージがさらに膵島、β細胞の小さなクラスター（&lt;10細胞）のディストリビューション全体が含まれています。 <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="図1b" /> そのまま成人の膵臓のβ細胞の全体的な分布の図1B仮想スライスの画像解析 右側の背側膵と左の腹側膵と統合された仮想スライス。スケールバーは5 mmである。 代表的な結果バーチャルスライスイメージングのための膵臓の熟練準備ははっきりと異なる島で仮想スライス画像全体膵臓内のすべてのGFPタグ付きβ-細胞の効果的な定量化が可能になります。 その場で膵臓全体の成功バーチャルスライス画像も同様にβ-細胞のみならず、個々と全体の膵島面積だけでなく、膵島のサイズ分布、地域分析、および成長パターンを調べ、定量化する手段を提供します。 ImageJは仮想スライス画像の分析で同時にすべてのβ-細胞を検出する能力を維持しながら、それはまた、特定の膵島のサイズ（面積または直径によって）に分析を制限することによって、または場所によって定量化するためのβ-細胞を制御することができます。標準分析では、アーティファクトの例、単一のβ-細胞（&lt;170μmの2）と、レコード領域、周囲（地域を取り巻く距離）、循環（1.0は完全な円を表す真円度、）よりも小さい破片を、除外と各島のためのフェレーの直径（エリア内の最も長い距離）が検出されました。仮想スライス分析は、その形状に応じて個々の小島に重なって島を区別し、分離流域セグメンテーションを採用することで、仮想スライス画像を向上させることが可能です。分析は、さらに詳細な一連のイメージを生成しますが、これはそれぞれ個別の番号と対応する情報（図中の表に記載されている低域と高強度（図1C）の両方の下の画像のマスク、すべての推定膵島の輪郭を、含まれています。1D）、そしてオリジナルの仮想スライス画像（図1B）。しきい値を選択する潜在的な技術的なバイアスが大規模な構造を囲む余計な光を含むことにより、または特に小島、小さな粒子からの微弱な信号の総数を減らすことによって、島のスキュー定量化できることに注意してください。 <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="図1c" /> そのまま成人の膵臓のβ細胞の全体的な分布の図1C仮想スライスの画像解析 仮想スライスの蛍光粒子のマスク。カラーコード：[1]青：低強度の蛍光、[2]緑：中間強度の蛍光、及び[3]赤：高輝度の蛍光。 <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="図1d" /> そのまま成人の膵臓のβ細胞の全体的な分布の1次元仮想スライスの画像解析を図 個々の島/β-細胞のクラスターの定量化。各島が（β-細胞の小さなクラスターを含む）対応するグラフで説明するその情報を、番号付けされていることに注意してください。 （3）真円度、（2）境界、（1）面積：：4つのパラメータは、それぞれの構造のために採取した数1.0は完全な円を表す丸みの程度、（4）フェレーの直径：エリア内で最長の距離。 ＃706はわずか数β-細胞の面積と分析結果の解像度を表示することに注意してください。流域セグメンテーションなど示されているものとして、隣接する島のグループを検出し、適切に明確な小島（小島1554、1555、および1556）として、それらを区別します。 <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="図1e" /> そのまま成人の膵臓のβ細胞の全体的な分布の図1E仮想スライスの画像解析 面積、境界、およびフェレーの直径の軸を持つ仮想スライスの解析の3次元プロット、。各ドットは、膵島を表します。

<ol>
	<li>Miller, K., Kim, A., Kilimnik, G., Moka, U., Jo, J., Periwal, V., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+formation+during+the+neonatal+development+in+mice.">Islet formation during the neonatal development in mice.</a> PloS One. 4, E7739-E7739 (2009).</li><li>Kilimnik, G., Kim, A., Jo, J., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+pancreatic+islet+distribution+in+situ+in+mice.">Quantification of pancreatic islet distribution in situ in mice.</a> Am J Physiol Endocrinol Metab. 297, E1331-E1338 (2009).</li><li>Hara, M., Wang, X., Kawamura, T., Bindokas, V. P., Dizon, R. F., Alcoser, S. Y., Magnuson, M. A., Bell, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells.">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> Am J Physiol Endocrinol Metab. 284, E177-E183 (2003).</li><li>Hara, M., Dizon, R. F., Glick, B. S., Lee, C. S., Kaestner, K. H., Piston, D. W., Bindokas, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pancreatic+beta-cells+in+the+intact+pancreas.">Imaging pancreatic beta-cells in the intact pancreas.</a> Am J Physiol Endocrinol Metab. 290, E1041-E1047 (2006).</li></ol>

利害の衝突は宣言されません。

無傷の膵臓のランゲルハンス島とβ細胞のクラスターの仮想スライス画像の解析は、島の全体の分布の大規模なビューを提供します。発展と時間をかけて合計膵島の分布の変化は、このように研究し、比較することができます。そのような例は、膵島の形成の分析（1）で示されています。様々な時点（P1 - P21）での新生児マウスの膵臓の包括的な分析は、島が分裂によって形成されることが?...

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<td>Forceps</td>
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<td>Electron Microscopy Sciences</td>
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in situ quantification of pancreatic beta-cell mass in mice

健康と病気の特定の細胞集団の変化をトレースすると生物医学研究の重要な目標です。モニタリングの膵臓β細胞の増殖および膵島の成長のプロセスは特に困難である。我々はImageJ（rsb.info.nih.gov / IJ /）用に書かれたマクロを使用して蛍光タグ付きのβ-細胞とトランスジェニックマウスの無傷の膵臓のβ細胞の分布をキャプチャする方法を開発した。膵臓の解剖と組織の清算に続いて、全体の膵臓は、その後GFP -タグ付きβ-細胞が検討されている、仮想スライスとしてキャプチャされます。分析は、各島のためとβ-細胞の小さなクラスターのための特定のパラメータとの位置への参照を使用して、全β-細胞の面積、膵島の数とサイズ分布の定量化が含まれています。島の全体的な分布は、3次元にプロットし、各島のサイズと形状分布からの情報が一目で全体的なβ細胞面積の変化の定量的および定性的な比較を可能にすることができます。

Watch this Scientific Journal Video about In situ Quantification of Pancreatic Beta-cell Mass in Mice at JoVE.com

In situ Quantification of Pancreatic Beta-cell Mass in Mice

以下のプロトコールは、膵臓の仮想スライスイメージングのための解剖、および全体の膵臓内のすべてのGFPタグ付きβ-細胞のその後の定量化のプロセスを概説します。

マウスにおける膵β細胞の質量のその場定量に

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

in-situ-quantification-of-pancreatic-beta-cell-mass-in-mice

Research

JoVE Journal

Biology

In situ Quantification of Pancreatic Beta-cell Mass in Mice

12.1K ビュー.  University of Chicago. 以下のプロトコールは、膵臓の仮想スライスイメージングのための解剖、および全体の膵臓内のすべてのGFPタグ付きβ-細胞のその後の定量化のプロセスを概説します。

ビデオ: In situ Quantification of Pancreatic Beta-cell Mass in Mice

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

糖尿病マウスの腎臓のカプセルに膵島の移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

生物学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

膵島質量、サイズ分布とアーキテクチャのコンピュータ支援大規模可視化と定量化

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

質量分析法とあいまってペプチドイムノエンリッチメントを使用したタンパク質の定量化

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

長指伸筋の筋機能の評価<em&gt;エクスビボ</em&gt;と前脛骨筋<em&gt;その場</em&gt;マウスの

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

結晶セルロース蓄積中の高解像度の定量化<em&gt;シロイヌナズナ</em&gt;ルーツは、組織特異的な細胞壁の変更を監視します

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

蛍光を用いて観察し、テロメアの定量化と反復配列<em&gt;その場で</emPNAプローブのある&gt;ハイブリダイゼーション（FISH）<em&gt;線虫（Caenorhabditis elegans）</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Methods Article

マウスにおける膵β細胞の質量のその場定量に