この作品は、老化の国立研究所（AG04418）、国立薬物乱用研究所、健康、カロリンスカ研究所大学院のパートナーシッププログラムの国立研究所、カロリンスカ研究所、スウェーデン研究評議会、スウェーデンの頭脳パワー、およびスウェーデンの脳の財団によってサポートされていました。多くの、それぞれ図1と2、創造的サポートのためのマティアスカーレンと博士ジュゼッペCoppotelliのおかげで、技術支援のためのPernoldカリン、と博士。バリーJ.ホッファー、非常に有益な助言と議論するためのラースオルソン、そしてニルス-ゴランラーション。

1。 cryosectioningのための組織の準備<ol><li>利用可能な倫理的な許可証に基づき、頸椎脱臼または断頭のいずれかの方法で動物を生け贄に捧げる。 </li><li>迅速に関心の組織（ 例えば 、脳）を収集し、迅速に（組織の最適な形態を得るために液体窒素で冷やしイソペンタンまたはプロパンで凍結が必要になる場合があります）ドライアイスで凍結。セクションに-80℃アルミホイルで店の組織は、° Cまでの準備。 </li><li> cryosectioningの準備のために凍結組織を埋め込みます。 </li><li> -21で14μmの凍結切片を収集° C（温度を調整する必要があります± 1〜2 ° C）。使用する準備ができるまで-20℃で封入することなく加熱ブロック、および店舗のスライドを使用してスライド上のセクションを解凍します。 </li></ol> 2。 COXの組織化学<ol><li>スライドは、1時間室温で乾燥することができます。湿った濾紙を持つスライド染色室でスライドを入れ、ストリップに切断。 consistenを取得するには各実験におけるtの結果、それは時間の遅延を最小限に抑えるため、実験ごとに10個のスライドの最大値を処理することをお勧めします。 </li><li>化学フード1X DAB、0.1 M PBSのpH = 7.0で100μM シトクロム cの下準備。すぐに渦。 </li><li> （2μgのml -1のまたは約4 IU ml -1の ）カタラーゼ、ウシ2μgのを追加。カタラーゼのすべての穀物を分割してボルテックスでよく混和する。 </li><li> 150適用 - インキュベーション培地200μLを各スライドに、すべてのセクションの上に均等に分散するためにピペットチップを使用してください。 </li><li> 37℃で40分間スライドをインキュベート℃に</li><li>スライド上の過剰な溶液を除去。洗浄は、0.1 M PBSのpH = 7.0の4倍、10分ごとに時間を、スライド。 </li><li>湿紙のストリップをスライド染色室にスライドを返します。 </li></ol> 3。 SDHの組織化学<ol><li> 1.5mMのNBT、130mMのコハク酸ナトリウム、0.2mMのPMS、および0.1MのPBSのpH = 7.0の1.0 mMのアジ化ナトリウムは、化学物質のボンネットの下準備。遮蔽するために十分注意してください光からPMS。すぐに渦。 </li><li>各スライドにインキュベーション培地の150〜200μLを適用し、すべてのセクションの上に均等に分散するためにピペットチップを使用してください。 </li><li> 37℃で40分間スライドをインキュベート℃に</li><li>スライド上の過剰な溶液を除去。洗浄は、0.1 M PBSのpH = 7.0の4倍、10分ごとに時間を、スライド。 </li><li>エタノールの下記の濃度で2分間スライドを脱水：70％、70％、95％、95％、99.5％。その後は、追加の99.5％の段階で10分の時間を要します。 </li><li> 10分間、キシレンのスライドを置きます。 Entellanとカバースリップをマウントします。スライドは、換気の良い場所で一晩、または少なくとも1〜2時間乾燥することができます。 </li></ol> 4。ミトコンドリア機能障害の定量<ol><li>ミトコンドリア機能障害の量は、細胞ブルー染色の量で示されます。これらの金額を半定量するために、スライドはコード化し、明視野顕微鏡下で可視化されるべきである。半定量は、ブラインドで実行する必要があります<html>スケールを使用して基礎は、0 - 4（0、青なし染色、4、唯一青色の染色）から、例えば。各被験者/動物の平均値を計算するために与えられた件名（subject）/動物からいくつかのセクションで半定量化のこの種類を実行するのが最善です。 </li><li>統計は、マン - ホイットニーまたはKruskal - Wallisのような非パラメトリックテストを、使用して実行する必要があります。 </li></ol> 5。適切な特異性のコントロール<ol><li> COX活性のための特異性のコントロールについては、&quot;COXの組織化学&quot;の手順を繰り返し、および2.5 mMのアジ化ナトリウム、ターミナルの呼吸鎖阻害剤を加える。 </li><li> SDH活性のための特異性のコントロールについては、コハク酸ナトリウムの除去し、50mMマロン酸、SDHの競合阻害剤を加えて&quot;SDHの組織化学&quot;の手順を繰り返します。 </li><li>洗い、エタノール系列でセクションを脱水してから、などの手順3.4で説明したスライドにマウントし、カバースリップ - 3.6。 </li></ol> 6。代表的な結果： テント&quot;&gt; COX / SDH二重標識組織化学的アッセイの全体的なスキームを図2に示されています。野生型と年不相応に老けているmtDNAの突然変異誘発のマウスからの脳切片における適切なCOX / SDH二重標識組織化学の代表的な例が図に示されている（3）野生型マウス（図3、左パネル）の暗褐色の染色は、通常のCOXの活性を示した。青色染色によって示される呼吸鎖の欠陥を持つ細胞は、12週齢のミトコンドリアDNAの突然変異誘発のマウスでは明らかに、これらの欠陥は、となった46週間（図3、中央と右のパネル）に歳のmtDNAミューテータマウスとして普及。 不十分なCOXラベリングのために野生型マウスの脳切片における不適切なCOX / SDH二重標識の例を図4に示されています。不十分なインキュベーションのCOX活性のデモンストレーションのための時間、またはインキュベーション中にスライドを封入により、分子状酸素の利用を減らすことは、DAB反応するの低下沈着をもたらしたしたがって、イオン積、とは、SDHのインキュベーション中に、青色のホルマザンの最終製品の形成を可能にした。 COXとSDH活性はまた（図5、左と中央）別々に調べることができますが、系列ラベリングには、COXの不足で細胞の同定に有用であり、SDHのインキュベーション（図3中の青い沈殿が形成されるため、中心をと右）。 COXおよびSDH活動のための特異性のコントロールも（図5、右）に行うことができます。 <img alt="図1" src="/files/ftp_upload/3266/3266fig1.jpg" /> 図1。ミトコンドリア呼吸複合体IV。ミトコンドリア呼吸鎖は、内膜内に位置し、5つの複合体を含んでいます。呼吸鎖の目的は、複雑なIからIVにして、ATPを生産する複合V（ATPアーゼ）で使用されている内側の膜を横切るプロトン勾配を作るそうすることで電子を輸送することです。赤い六角形represeNTのサブユニットはミトコンドリアDNAによってコードさ。白い六角形は、核DNA（複合体IIが完全に核ゲノムからエンコードされていることに注意）でエンコードされたサブユニットを表しています。従って、ミトコンドリアゲノムの変異は、呼吸鎖複合体のサブユニットの変異に起因する呼吸鎖の機能障害を引き起こす可能性があります。 <img alt="図2" src="/files/ftp_upload/3266/3266fig2.jpg" /> 図2。COX / SDH二重標識組織化学アッセイのフローチャート 。興味の器官を分析、急速にドライアイス上で組織を凍結し、そして、それらを保存する - 80℃をクリオスタット切片を収集し備え置かなければならない - 20 ° Cまでの使用を。 1時間室温で空気乾燥のセクションを許可する。 、COXの組織化学のためのインキュベーション培地を準備したスライドに適用し、37℃で40分間インキュベート℃を10分各洗浄用PBS 4倍のセクションを洗ってください。 SDHの組織化学のために培養培地を調製、SLIに適用DESは、再度37℃で40分間インキュベートPBSでもう一度セクションを洗浄、エタノール系列で脱水し、マウントしてスライドをカバースリップ。 COX / SDH二重標識のセクションでは、1〜2時間以内に明視野顕微鏡下で表示する準備ができました。 <img alt="図3" src="/files/ftp_upload/3266/3266fig3.jpg" /> 図3。COX / SDH二重標識の代表例 。野生型と年不相応に老けているmtDNAの突然変異誘発のマウスの脳切片を順次COXおよびSDH活動のために標識した。 （スケールバー：200μmの）通常のCOX活性は、（ダークブラウン色で示される）野生型マウス（左）から海馬に示す通りであった。 COXの欠陥は、（青い色で示される）のmtDNAミューテータマウス（中央と右）から海馬において明らかにされた。 mtDNAの突然変異誘発マウスの年齢の46週までにCOX活性のさらなる減少は、呼吸鎖の機能障害の広範な悪化を示唆し、そこにあった。観測された水戸mtDNAの突然変異誘発マウス12のchondrial機能障害は、ミトコンドリアDNAの点突然変異だけでなく、リニア失5の増加したレベルの高レベルによって引き起こされる。 <img alt="図4" src="/files/ftp_upload/3266/3266fig4.jpg" /> 図4。不適切なCOX / SDH二重標識の例 。野生型マウスの脳切片を順次COXおよびSDH活動のために標識した。 （スケールバー：200μmの）不適切なインキュベーション時間（10〜25分）COX活性のデモンストレーションのためには、40分間のインキュベーション時間（左と中央）と比較して、茶色のDABの反応生成物の削減堆積をもたらした。 SDHのインキュベーション中に、青色のホルマザンの最終製品を形成するために許可されて短縮されたインキュベーション時間は、誤解を招くCOXの欠陥を持つ細胞の存在を示唆している。 COXのインキュベーション中にスライドを封入することも、DAB反応するの不正確な形成と沈着をもたらしたイオン製品（右）。 <img alt="図5" src="/files/ftp_upload/3266/3266fig5.jpg" /> 図5。私ndividual COXおよびSDHのラベリングと特異性の制御 。野生型マウスの脳切片を別々にCOXとSDH活動のために標識した、（左と中央）がそれぞれ、ダークブラウン色とブルー色で示されます。 COXおよびSDHの活動が個々にラベルを付けることができますが、系列ラベリングは、ミトコンドリア機能障害を有する細胞を見つけるのに有利であることが証明されている。野生型マウスから脳内COXおよびSDH活動のための特異性の制御の例は、ラベルの有無を（右）を示した。 （スケールバー：200μmの）

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+tRNA(Leu(UUR))+mutation+in+mitochondrial+encephalomyopathy,+lactic+acidosis,+and+strokelike+episodes+(MELAS):+genetic,+biochemical,+and+morphological+correlations+in+skeletal+muscle.">The mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic, biochemical, and morphological correlations in skeletal muscle.</a> Am. J. Hum. Genet. 50, 934-949 (1992).</li><li>Petruzzella, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremely+high+levels+of+mutant+mtDNAs+co-localize+with+cytochrome+c+oxidase-negative+ragged-red+fibers+in+patients+harboring+a+point+mutation+at+nt+3243.">Extremely high levels of mutant mtDNAs co-localize with cytochrome c oxidase-negative ragged-red fibers in patients harboring a point mutation at nt 3243.</a> Hum. Mol. Genet. 3, 449-454 (1994).</li><li>Tulinius, M. H., Holme, E., Kristiansson, B., Larsson, N. G., Oldfors, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+encephalomyopathies+in+childhood.+I.+Biochemical+and+morphologic+investigations.">Mitochondrial encephalomyopathies in childhood. I. Biochemical and morphologic investigations.</a> J. Pediatr. 119, 242-250 (1991).</li><li>Haas, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in-depth+evaluation+of+suspected+mitochondrial+disease.">The in-depth evaluation of suspected mitochondrial disease.</a> Mol. Genet. Metab. 94, 16-37 (2008).</li></ol>

利害の衝突は宣言されません。

複合COX / SDH組織化学的方法では、ミトコンドリア機能障害を有する細胞の可視化を可能にします。この手法は、初期の研究、1968年にさかのぼると、多くはそれの患者14,19,26,27のミトコンドリア病を識別するための&quot;ゴールドスタンダード&quot;を考慮して、人気を保っています。それは今頻繁にmtDNA変異主導老化や老化関連疾患12,13,18,20,21,24を調査するために使用されます。 C...

<table border="1"><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td>ドライアイス</td><td> AGAガスAB </td><td>ブロックの形</td><td></td></tr><tr><td>イソペンタン（2 - メチルブタン） </td><td>シグマアルドリッチ</td><td> 277258 CAS：78-78-4 </td><td></td></tr><tr><td> Cyrostat埋め込みソリューション</td><td>サクラファインテック</td><td>組織テック4583 </td><td></td></tr><tr><td>クライオスタット</td><td> Microm </td><td> MicromモデルHM 500M </td><td></td></tr><tr><td>スライド</td><td>サーモサイエンティフィック</td><td>スーパーフロストプラス メンツェルグレイザー J1800AMWZ </td><td></td></tr><tr><td>カバーガラス ホウケイ酸ガラス</td><td> VWRインターナショナル</td><td> 16004-098 </td><td> 24 × 50ミリメートル</td></tr><tr><td>ろ紙</td><td>テルフィルターAB </td><td>品質：1350 記事番号：242 001 </td><td> 430 × 430ミリメートル</td></tr><tr><td> 3,3&#39; - ジアミノベンジジン四塩酸塩（DAB） </td><td>シグマアルドリッチ</td><td>シグマ液体基質システム、D7304 </td><td></td></tr><tr><td> シトクロム c（タイプIII、ウマ心臓から） </td><td>シグマアルドリッチ</td><td> C2506 CAS：9007-43-6 </td><td></td></tr><tr><td> （肝臓から）カタラーゼ、ウシ</td><td>シグマアルドリッチ</td><td> C9322 CAS：9001-05-2 </td><td></td></tr><tr><td>ニトロブルーテトラゾリウム（NBT） </td><td>シグマアルドリッチ</td><td> N6876 CAS：298-83-9 </td><td></td></tr><tr><td>コハク酸ナトリウム</td><td>シグマアルドリッチ</td><td> S2378 CAS：6106-21-4 </td><td></td></tr><tr><td>フェナジンメトサルフェート（PMS） </td><td>シグマアルドリッチ</td><td> P9625 CAS：299-11-6 </td><td> PMSは、光に敏感です。光の盾。 </td></tr><tr><td>アジ化ナトリウム</td><td>シグマアルドリッチ</td><td> S8032 CAS：26628-22-8 </td><td></td></tr><tR&gt; <td>キシレン</td><td> VWRインターナショナル</td><td> EM - XX0060 - 4 </td><td></td></tr><tr><td> Entellan </td><td> VWRインターナショナル</td><td> 100503-870 </td><td></td></tr><tr><td>マロン酸エステル （マロン酸） </td><td>シグマアルドリッチ</td><td> M1296 CAS：141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

ミトコンドリアDNA（mtDNA）の欠陥は、病気の重要な原因であり、変化の1,2の高齢化や老化に関連する根底にある。老化のミトコンドリア説は、老化プロセス3で、生体エネルギーの恒常性と細胞機能を変化させることができるミトコンドリアDNA変異、の役割を示唆している。豊富な証拠は、この理論の1,4、mtDNAの突然変異誘発マウス5にする例のサポートでコンパイルされているが、高齢化のmtDNAの損傷の正確な役割は、6,7完全には理解されていない。 呼吸酵素の活性を観察することによりミトコンドリアの機能障害を調査するための簡単​​なアプローチです。複雑なIV、またはシトクロム c オキシダーゼ （COX）は、ミトコンドリアの機能に不可欠です。 COXの触媒サブユニットはミトコンドリアDNAによってエンコードされ、複合体の構築（図1）のために必須であるされています。従って、適切な合成と機能は、主にミトコンドリアDNAの完全性2に基づいています。他の呼吸器複合体を調査することもできますが、複合体IVとIIは、組織化学的検査8,9に最も適しています。増加はミトコンドリアの生合成10-12を示すかもしれませんが、複雑なII、またはコハク酸脱水素酵素（SDH）は、完全に核DNA（図1）によってエンコードされ、その活性は、通常、障害のあるミトコンドリアDNAの影響を受けません。障害ミトコンドリア病で観察されるミトコンドリア、老化、そして加齢に伴う病気は、しばしば低いか不在のCOX活性2,12-14と細胞の存在につながる。 COXおよびSDHの活動を個別に調査することができますが、連続した二重標識法15,16は、ミトコンドリア機能障害12,17-21で細胞を見つけるのに有利で ​​あることが証明されている。 アッセイの最適な憲法の多くは、基質濃度、電子受容体/供与体、中間の電子キャリア、pHの影響、及び反応のtとして、決定されているIME 9,22,23。 3,3&#39; -ジアミノベンジジン（DAB）は、効果的かつ信頼性の高い電子供与体22です。機能COXを持つ細胞では、茶色のインダミンポリマー生成物は、ミトコンドリアのクリステに局在し、細胞22を飽和します。機能不全にCOXとそれらの細胞は、青色ホルマザンの最終製品9,24に、ニトロブルーテトラゾリウム（NBT）、電子受容体の減少により、SDH活性の可視化を可能にする、DABの製品で飽和されることはありません。 シトクロム cとコハク酸ナトリウムの基板は、制御および変異型/疾患組織から9までの内因性レベルを標準化するために追加されます。カタラーゼペルオキシダーゼ活性9,22から可能な限り汚染反応を避けるための予防措置として追加されます。フェナジンメトサルフェート（PMS）、中間電子伝達体は、最終の反応生成物9,25の形成を高めるために、呼吸鎖阻害剤、アジ化ナトリウムと組み合わせて使用されます。これは、通知にもかかわらずationは、この上品な簡単なアッセイの結果に影響を及ぼすいくつかの重要な詳細は、特異性のコントロールやテクニックの進歩に加えて、まだ提示されていない。

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

シトクロムcオキシダーゼ/ナトリウム脱水素酵素（COX / SDH）二重標識法では、新鮮凍結組織切片におけるミトコンドリアの呼吸酵素の欠乏の直接可視化することができます。これは簡単な組織化学的手法であり、ミトコンドリア病、老化、および老化関連疾患を検討する上で有用です。

シトクロムを使用してミトコンドリア呼吸機能の可視化 Cオキシダーゼ/コハク酸脱水素酵素（COX / SDH）二重標識組織化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

visualization-mitochondrial-respiratory-function-using-cytochrome-c

Research

JoVE Journal

Biology

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

36.5K ビュー.  Karolinska Institutet. シトクロムcオキシダーゼ/ナトリウム脱水素酵素（COX / SDH）二重標識法では、新鮮凍結組織切片におけるミトコンドリアの呼吸酵素の欠乏の直接可視化することができます。これは簡単な組織化学的手法であり、ミトコンドリア病、老化、および老化関連疾患を検討する上で有用です。

ビデオ: Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

マイクロインジェクションを使用して安定したトランスジェニック線虫の世代

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

ザ ex vivoで</em血管反応性の調査のために>隔離された骨格微小血管の準備

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

腎細胞内のミトコンドリアの機能と細胞生存率の評価を過剰発現するプロテインキナーゼCアイソザイム

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

SOX10中の顔面の開発の可視化：楓トランスジェニックゼブラフィッシュラインタイムラプス共焦点顕微鏡を用いて

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

大規模な遺伝子ノックダウン中<em&gt; C.虫</em&gt;堅牢な機能喪失表現型を生成するためのdsRNA摂食ライブラリの使用

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

拘束されていない全身プレチスモグラフィを使用したマウスにおける呼吸機能の測定

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

透過処理と無傷の細胞でミトコンドリア機能を評価するために高解像度呼吸計測

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

ミトコンドリアの構造と機能の研究に<em&gt;ショウジョウバエ</em&gt;卵巣

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.