本研究は、ポスドクの中国特別基金からの助成金によって支えられた (YF に号 2014T70392)、中国の国家自然科学基金 (YF に号 81673766)、新しい教師プライミング基金、復旦大学、Zuoxue 財団、開発上海ピーク分野統合医学 (20150407) のプロジェクト。

動物を対象とするすべてのプロシージャ上海医科大学、復旦大学 (承認番号 20160225-013) の動物倫理委員会のガイドラインに従った。
1. 透明なマウス卵巣の準備
<ol><li>
溶液の調製
	<ol>
<li>リン酸緩衝生理食塩水 (PBS) ソリューション (1 M、pH 7.6) 0.1% 準備トリトン X-100 (pbst;)。10 x PBS 原液 1 リットルを作るためには、87 g の塩化ナトリウム、NaH2PO4 · 3.1 g を混ぜる2 H2O、および Na2HPO4 ·12H2O. 28.7 g dH2O に 1 L 攪拌 ~ 2 h. 調整 pH 7.4 に 1 N naoh を追加し、常温で保存します。1/10 この 10 倍原液を希釈 dH2O を使用し、0.1% トリトン X-100 PBS で。</li>
		<li>混合 (wt/巻) 4% パラホルムアルデヒド (PFA) ソリューション、4% (重量/巻) アクリルアミド、0.05% (重量/巻) bisacrylamide、ダブル蒸留水氷の上の 0.25% (重量/巻) VA 044 イニシエーターによるゲル溶液 (pH 7.5) を準備します。</li>
		<li>4% (重量/巻) ドデシル硫酸ナトリウム (SDS) 二重蒸留水に 200 mM ホウ酸ナトリウム緩衝を混ぜてクリア ソリューション (Ph8.5) を準備します。</li>
	</ol>
</li>
	<li>
Transcardial 血流と卵巣の準備
	<ol>
<li>すべてのプロシージャの間に氷の上のすべてのソリューションをしてください。</li>
		<li>ペントバルビ タール (0.35 mg/kg) ソリューションの腹腔内注入成人女性野生型 c57bl/6 マウスを麻酔します。それはつま先ピンチの応答を示しています、一度マウスに麻酔がかかる適切。</li>
		<li>フラットの発泡ボードを伸ばして手足でマウスを固定する粘着テープを使用。</li>
		<li>ハサミで胸、肌や骨を剣状突起上を切開すること、心臓を公開します。</li>
		<li>動物の右房切開を行い、20 ml 10ml/分で冷たい 1 × PBS の左心室を灌流します。</li>
		<li>少なくとも 20 ml ゲル液 10 mL/分での動物を灌流します。</li>
		<li>ハサミには、全体の enterocoelia を公開している medioventral ラインに沿って腹部の皮膚を切開し、腸を削除します。卵巣の形態の整合性を維持するために子宮と卵巣を収集します。</li>
		<li>顕微鏡下で卵巣の周りの脂肪組織を削除します。</li>
	</ol>
</li>
	<li>
脱ガス
	<ol>
<li>3 日間の固定のための 4 ° C で 10 mL ゲル ソリューションに卵巣を配置します。</li>
		<li>5 mL チューブに卵巣を転送し、ハイドロゲル ソリューションとチューブを入力します。チューブの上にパラフィルムをストレッチし、管の首にパラフィルムをラップします。 注意: は、パラフィルムとハイドロゲル ソリューションの泡がないことを確認します。</li>
		<li>37 ° C で重合するハイドロゲルを許可する 3 h の最大のためのインキュベーターのシェーカーにチューブを置きます。</li>
		<li>ヘラを使用してゲルから卵巣を削除、ティッシュ ペーパーで卵巣の周りから余分なゲルを注意深く取り外します。1x PBS で 4 回卵巣を洗います。</li>
	</ol>
</li>
	<li>
パッシブ クリア
	<ol>
<li>クリア溶液 10 mL で卵巣が水没します。消去プロセスを開始する 37 ° C で 80 rpm で軽く振る。</li>
		<li>最初の週に 3 日おきにソリューションを変更します。卵巣まで週が明確になったら、一週間後クリア ソリューションを更新します。通常このプロセスは、約 2-3 週間かかります。 注: 卵巣 immunostained を直接することができます。 またはに格納できるアジ化ナトリウム (0.02%) PBS の 4 ° C で免疫染色の前に数週間。</li>
	</ol>
</li>
</ol>2. 免疫染色
<ol><li>Pbst; で 2 回シェーカーで 37 ° C で 1 日目に卵巣を洗います。</li>
	<li>37 ° C で 2-3 日目の主な抗体/PBST ソリューション (表 1) にシェーカーで卵巣を孵化させなさい血小板内皮細胞接着分子 1 (CD31) をされた一次抗体をここで提示する仕事でチロシン水酸化酵素それぞれ 1:10 と 1:50 PBST で希釈します。 注意: 一次抗体の 2 つ以上の種類を使用している場合は、別の動物の源があるを確認します。</li>
	<li>シェーカーで 4 日目に 37 ° C で PBST バッファーと一次抗体を洗浄します。</li>
	<li>シェーカーで 37 ° C で 5 - 6 日目 PBST 目的の二次抗体と卵巣を孵化させなさい。ここで紹介する作業、二次抗体はチロシン水酸化酵素と 1:50 PBST 内で希釈した CD31 の Alexa 小麦粉 594 が Alexa 小麦粉 488 とだった。</li>
	<li>シェーカーで 7 日で 37 ° C で PBST バッファーと二次抗体を洗浄します。</li>
	<li>屈折率マッチング ソリューション 8 日目に屈折率の均質化のために組織を転送します。目盛線目盛線を見ることができるかどうかを確認することでその映像の明瞭さをチェックすると紙の上には、組織を置きます。組織が完全に明らかにされて、一度はイメージすることができます。</li>
	<li>作ることをちょうど囲むパテ シリンダー、組織とし馬蹄形に形状、密閉された空間を作るためにスライド ガラスにしっかりと外縁隆起帯を押します。パテの高さは卵巣より少し背が高いはずです。</li>
	<li>パテの中心に明らかに卵巣を慎重に配置し、屈折率整合ソリューションを追加します。しっかりとパテの上にガラス底皿を置くし、お皿を修正するためパテを使用します。</li>
</ol>3. 共焦点顕微鏡
<ol><li>共焦点顕微鏡を用いたイメージング、「Ni E」パネルで (この場合 2.0 mm) の適切な作動距離で (この場合は水浸漬 25 X 対物レンズ、1.1 NA、2 mm WD) レンズを選択します。</li>
	<li>
「取得」パネルでチャンネル数を選択します。ここでは、3 つのチャンネルを使用します。
	<ol>
<li>まず、顕微鏡接眼レンズを通じてチェックすることによって組織をフィールドの中央に移動するのに「目ポート」タブと XY コント ローラー ジョイスティックを使用します。</li>
		<li>シャッター光を切って"HV (利得)"、「レーザー パワー」を調整する「ライブ」のタブをクリックし、"オフセット ' 各チャネル個別に。ハイパワー レーザーおよび HV より高い信号をもたらすし、高いオフセットがバック グラウンド ノイズを減らすことができます。</li>
		<li>適切な「スキャン サイズ」と「数」を選択します。スキャン サイズを 1024 x 1024 μ m2 4 ~ 8 のカウントを使用します。</li>
	</ol>
</li>
	<li>
「Z 強度補正」パネルで組織の深さを定義する画像品質を設定後、(で、組織の中心) 卵巣の最上位層のレンズを見つけるとトップ層の獲得を調整後、「プラス」をクリックトップにレイヤーを定義します。
	<ol>
<li>レンズを下に移動、適切な HV と組織の下のレーザー パワーを設定し、Z 強度補正テーブルに最低のレイヤー情報を追加します。「ND 取得」パネルで設定を同期するの"に ND"をクリックします。</li>
	</ol>
</li>
	<li>
「ND 取得」パネル、スキャン数のステップの最初のセット。その後、"Z"と「大きな画像」タブを選択します。
	<ol>
<li>接眼レンズを通して組織の可視化の助けを借りて、組織の境界線に従ってフィールドのサイズを定義する「大きな画像をスキャン」ウィンドウに移動します。スキャン領域 (「フィールド」) のサイズを入力し、10% と 15% 間の重複を選択して「ND 取得」パネルに戻ります。</li>
		<li>「実行 Z 補正」、ない「今すぐ実行」自動的に各レイヤーに適切な HV、レーザー出力とオフセットを設定し、画像を処理するのを待つをクリックしてします。</li>
	</ol>
</li>
</ol>4. 個別包とその血管の三次元
<ol><li>まず、ImageJ5を使用して、元の NIS ファイルを開きます。「イメージ」ボタンをクリックしてし、「色」と"分割チャンネル"を選択します。「ファイル」をクリックし、「名前を付けて保存」で「イメージ シーケンス」オプションを使用して tiff ファイル シリーズとしてチャンネルを個別に保存します。</li>
	<li>Imaris を使用すると、tiff ファイル シリーズのうちの 1 つを開きます。「編集」ボタンをクリックし、「チャンネルの追加」を選択してチャンネル残り部分を追加します。「ディスプレイの調整」タブでは、名前およびチャネルの色を変更できます。 されます組織の厚さは、「編集」下のドロップ ダウン リストで「イメージ プロパティ」パネルで修正される必要があります。</li>
	<li>最終のイメージと余分な部分の除去の 3 D のトリミング、"Edit"ボタンをクリックし、「トリミング 3 D」を選択します。フィールドのサイズは、枠線をドラッグして調整できます。</li>
	<li>
サーパス パネルにおけるフィラメント アルゴリズムを用いた船舶信号を再構築するには、新しいフィラメントを作成し、次へをクリックする「フィラメント」をクリックします。
	<ol>
<li>最大かつ最薄の直径を定義するために「スライス」パネルに移動しをトレース船を見つけます。容器の最大幅で 2 つの点を選択することにより、距離が自動的に「測定」パネルに表示されます。</li>
		<li>「サーパス」パネルに戻ると幅の測定値を入力次へをクリックして。</li>
	</ol>
</li>
	<li>開始とシード ポイントのしきい値を調整します。「カメラ」パネルで「選択」を選択します。自動的に作り出された球のいくつかが削除する場合は、shift キーを押し、ポイントをクリックします。「移動」を選択して、マウスを動かして、によってすべての正しいポイントが保持されている, し、次へをクリックを確認する構造を回転できます。</li>
	<li>局所的なコントラストの高いしきい値を選択し、[次へ] をクリックします。「検出の棘」を選択しないし、血管柱の再構成を完了する続行。色は、「カラー」タブをクリックしてして編集できます。再建されたフィラメントの「統計」パネルで統計データを抽出できます。[編集] パネルで余分な部分を削除できます。 注: 合計卵胞または濾胞壁のボリュームを測定するための他のアルゴリズムを使用できます。ここで紹介する作業、卵母細胞は両方の二次抗体の蛍光によって示されました。データ量が大きいためワークステーションのサーバーは、データ分析のため使用されました。</li>
</ol>5. 統計解析
注: ± 標準誤差を手段として、画像解析データが掲載されています。
<ol><li>独立したサンプルの t 検定を使用して卵胞の比較実行し、ピアソンの相関テストを使用して卵胞壁毛細血管の長さの相関係数を比較します。P の値設定 < 統計的有意性の制限と 0.05 です。</li>
</ol>

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U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Erturk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Kuwajima, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clear(T):+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">Clear(T): a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140, 1364-1368 (2013).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Lai, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationalisation+and+validation+of+an+acrylamide-free+procedure+in+three-dimensional+histological+imaging.">Rationalisation and validation of an acrylamide-free procedure in three-dimensional histological imaging.</a> PLOS ONE. 11, e0158628 (2016).</li><li>Susaki, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Tainaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159, 911-924 (2014).</li><li>Liu, A. K. L., Lai, H. M., Chang, R. C. C., Gentleman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-of-acrylamide+SDS-based+tissue+clearing+(FASTClear):+A+novel+protocol+of+tissue+clearing+for+three-dimensional+visualisation+of+human+brain+tissues.">Free-of-acrylamide SDS-based tissue clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.</a> Neuropathol Appl Neurobiol. 43, 346-351 (2016).</li><li>Xu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+three-dimensional+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.</a> Sci Rep. 7, 9895 (2017).</li><li>Migone, F. F., Cowan, R. G., Williams, R. M., Gorse, K. J., Zipfel, W. R., Quirk, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+an+essential+role+of+vasoconstriction+in+rupture+of+the+ovarian+follicle+at+ovulation.">In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.</a> Proc Natl Acad Sci U S A. 113, 2294-2299 (2016).</li><li>Malki, S., Tharp, M. E., Bortvin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+whole-mount+approach+for+accurate+quantitative+and+spatial+assessment+of+fetal+oocyte+dynamics+in+mice.">A whole-mount approach for accurate quantitative and spatial assessment of fetal oocyte dynamics in mice.</a> Biol Reprod. 93 (113), (2015).</li></ol>

著者が明らかに何もありません。

現在の研究では、毛細血管と個々 の発育卵胞の間の関係を評価するイメージング 3 D を提案する.筆者らは同じプロトコル9を使用して、大血管、毛包とそのままマウス卵巣内の卵胞の場所間の相互作用の役割を検討しました。パッシブわかりやすくアプローチでは、マイクロおよびマクロ生、卵胞形成、および黄体と卵胞だけでなくさまざまな発達段階で卵巣の建築を再建?...

卵胞が卵巣の構造と機能の基本単位とその開発は、卵巣内にある血管に関連性の高い。血管は栄養と卵胞ホルモンと成長と1卵胞の成熟に重要な役割を担っています。
選択的血管マーカー、トランスジェニック マウス モデル、医薬品開発などの技術の組み合わせは、卵巣の血管網、血管新生、血管の機能に関する知識を増加しています。卵胞形成。卵巣は、卵胞発育と排卵の間に様々 な組織や血管のネットワークを改造するためにアクティブな器官と呼ばれます。サイズと血管の構造のようなアクティブな改造開発、採用に卵胞の生物学的機能に必要です。
卵巣のセクションおよび血管の反応を使用して従来の組織学的および組織計量方法は二次元 (2 D) 画像2に限定されます。三次元 (3 D) 再構成技術の発展に伴い、組織スライスの 2 D 画像を 3 D 構造に重ねることができますが、この方法はまだいくつかの制限を持って-微細構造の一部を破壊することが組織の区分、組織は、不足していると多くの労働力がスライスから得られた画像からの 3次元再構成の製作にかかわっています。全体組織 3 D 共焦点顕微鏡イメージングは、これらの制限の多数を克服できるがこれらのメソッドは萌芽期卵巣3における血管新生の評価に限定。生後および成体卵巣におけるこれらの問題を解決するために可視化されたボリュームを増やして全体ティッシュ クリア透明度4などのメソッドを使用して、このようなメソッドは任意の構造変形せず卵巣の光のクリアランスを提供します。そのまま卵巣の 3 D 構造のイメージングは、本作で使用されている Imaris ソフトウェア パッケージなどの画像解析ソフトウェアの正確な画像データベースを提供します。
動的な生理学的システムの一部である成人期にわたって卵巣の改造、これ卵巣血管新生の調節に調査のための優秀なモデル。さらに、多嚢胞性卵巣症候群、卵巣癌などの女性生殖器系の病的な状態で卵巣血管の役割の評価は全卵巣組織イメージングを通じて学ぶことができます。パッシブのクラリティ法の開発と高度な画像解析ソフトウェアの使用は、血管や毛包などの卵巣構造の関係に関する詳細な空間情報を提供しました。

<table><tbody><tr><td>Acrylamide</td><td>Vetec</td><td>v900845</td><td>http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td></tr><tr><td>Alexa Flour 488 (Dilution 1:50)&amp;nbsp;</td><td>Life Technologies</td><td>A11039</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td></tr><tr><td>Alexa Flour 594 (Dilution 1:50)</td><td>Life Technologies</td><td>A11012</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>Bisacrylamide</td><td>Amresco</td><td>172</td><td>http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td></tr><tr><td>Black wall glass bottom dish (Willco-Dish)</td><td>Ted Pella</td><td>14032</td><td>http://www.tedpella.com/section_html/706dish.htm#black_wall</td></tr><tr><td>Boric acid</td><td>Sinopharm Chemical Reagent</td><td>10004818</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td></tr><tr><td>Disodium hydrogen phosphate dodecahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 12H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>10020318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td></tr><tr><td>FocusClear</td><td>Celexplorer</td><td>FC-102</td><td>http://www.celexplorer.com/product_list.asp?MainType=107&amp;amp;BRDarea=1</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td>http://www.parafilm.com/products</td></tr><tr><td>Paraformaldehyde</td><td>Sinopharm Chemical Reagent</td><td>80096618</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td></tr><tr><td>PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td><td>Abcam</td><td>ab28364</td><td>http://www.abcam.com/cd31-antibody-ab28364.html</td></tr><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&amp;amp;region=US</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sinopharm Chemical Reagent</td><td>10019318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td></tr><tr><td>Sodium dihydrogen phosphate dihydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>20040718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sinopharm Chemical Reagent</td><td>30166428</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sinopharm Chemical Reagent</td><td>10019718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td></tr><tr><td>Triton X-100</td><td>Sinopharm Chemical Reagent</td><td>30188928</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td></tr><tr><td>Tyrosine hydroxylase (TH, Dilution 1:50)</td><td>Abcam</td><td>ab76442</td><td>http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td></tr></tbody></table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

卵巣は、女性の生殖器系の主要な器官と雌性の配偶子の生産構造の複雑な関係との三次元 (3 D) 血管アーキテクチャが、内分泌系を制御するために不可欠です、卵巣は記述されていません。3 D の接続とそのまま卵巣の血管の構造を視覚化するために最初の重要なステップは、光学的に透明の卵巣をすることです。ヒドロゲルを用いて組織の収縮を避けるためには、受動的な明快さの固定ベース (アクリルアミド ハイブリダイズ リジッドのオフ脂質交換イメージング/染色/の場-交配-互換性のある組織ハイドロゲル) プロトコルをそのまま卵巣をクリアする方法.染色、高度な多光子の共焦点顕微鏡と 3 D 画像再構成それから卵巣の血管と濾胞性毛細血管の可視化のために使用されました。このアプローチを使用すると、有意な正の相関を示した (P < 0.01) 卵胞の毛細血管と卵胞壁量の長さの間。

我々 は受動的な卵巣の卵胞期と血管のアーキテクチャを維持すると、容器と包のラベル付きのマーカーからの最高の蛍光信号の取得をオフに迅速かつ簡単な方法にパッシブのクラリティ法を適応しました。卵胞血管の 3 D アーキテクチャは、CD31、内皮細胞6マーカーの免疫染色により決定されました。CD31 染色成体マウスの卵巣では卵胞の毛細血管と...

Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

ここでそのままマウス卵巣における受動的な明快さと卵巣の血管と濾胞性毛細血管の可視化における 3 D 画像再構成法の適応を提案する.

受動的な明快さクリア マウス卵巣の血管の 3次元再構築

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

three-dimensional-reconstruction-vascular-architecture-passive

Research

JoVE Journal

Biology

8.6K ビュー.  Fudan University. ここでそのままマウス卵巣における受動的な明快さと卵巣の血管と濾胞性毛細血管の可視化における 3 D 画像再構成法の適応を提案する.

ビデオ: Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで</em腫瘍免疫応答の>腫瘍イメージングとモニタリング

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

免疫・感染学

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

発生生物学

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

「マウス卵巣における現象を可視化する卵巣の組織培養

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

イメージングは​​3DISCOにより、細胞レベルで無傷生物系をクリア

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

神経科学

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

ハイC：ゲノムの三次元構造を研究する方法。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

生物学

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

成人マウス卵巣における正確な卵胞列挙体

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

マウスの発達中の卵巣保護区の定量的3D分析のための全卵巣免疫蛍光、クリアリング、および多光子顕微鏡

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

生化学

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

受動的な明快さクリアマウス卵巣の血管の 3次元再構築

要約

さらに動画を探す

この動画の章

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで腫瘍イメージングとモニタリング

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

「マウス卵巣における現象を可視化する卵巣の組織培養

イメージングは3DISCOにより、細胞レベルで無傷生物系をクリア

ハイC：ゲノムの三次元構造を研究する方法。

成人マウス卵巣における正確な卵胞列挙体

マウスの発達中の卵巣保護区の定量的3D分析のための全卵巣免疫蛍光、クリアリング、および多光子顕微鏡

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

人工多能性幹細胞に由来するマウスの作製

受動的な明快さクリア マウス卵巣の血管の 3次元再構築

要約

さらに動画を探す

この動画の章

のための免疫担当マウスにおける漿液性卵巣癌の同所性モデル in vivoで腫瘍イメージングとモニタリング

三次元レンダリングとカルビンディン抗体、解析人間の胎盤絨毛血管のネットワークを明らかにしました。

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

「マウス卵巣における現象を可視化する卵巣の組織培養

イメージングは​​3DISCOにより、細胞レベルで無傷生物系をクリア

ハイC：ゲノムの三次元構造を研究する方法。

成人マウス卵巣における正確な卵胞列挙体

マウスの発達中の卵巣保護区の定量的3D分析のための全卵巣免疫蛍光、クリアリング、および多光子顕微鏡

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

人工多能性幹細胞に由来するマウスの作製

受動的な明快さクリアマウス卵巣の血管の 3次元再構築

イメージングは3DISCOにより、細胞レベルで無傷生物系をクリア