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 border="0" cellpadding="0" cellspacing="0" width="1664">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Acrylamide</td>
			<td style="width:189px;">Vetec</td>
			<td style="width:127px;">v900845</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 488 (Dilution 1:50)&#160;</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11039</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 594 (Dilution 1:50)</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11012</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bisacrylamide</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:127px;">172</td>
			<td style="width:861px;">http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Black wall glass bottom dish (Willco-Dish)</td>
			<td style="width:189px;">Ted Pella</td>
			<td style="width:127px;">14032</td>
			<td style="width:861px;">http://www.tedpella.com/section_html/706dish.htm#black_wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Boric acid</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10004818</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10020318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FocusClear</td>
			<td style="width:189px;">Celexplorer</td>
			<td style="width:127px;">FC-102</td>
			<td style="width:861px;">http://www.celexplorer.com/product_list.asp?MainType=107&#38;BRDarea=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Parafilm</td>
			<td style="width:189px;">Bemis</td>
			<td style="width:127px;">PM996</td>
			<td style="width:861px;">http://www.parafilm.com/products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Paraformaldehyde</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">80096618</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab28364</td>
			<td style="width:861px;">http://www.abcam.com/cd31-antibody-ab28364.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Photoinitiator VA044</td>
			<td style="width:189px;">Wako</td>
			<td style="width:127px;">va-044/225-02111</td>
			<td style="width:861px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium azide</td>
			<td style="width:189px;">Sigma</td>
			<td style="width:127px;">S2002</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">20040718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium dodecyl sulfate</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30166428</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium hydroxide (NaOH)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Triton X-100</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30188928</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Tyrosine hydroxylase (TH, Dilution 1:50)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab76442</td>
			<td style="width:861px;">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

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

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 ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

三次元蛍光画像から細胞形態の変化を定量分析ツール

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

精巣生殖細胞の三次元クロマチンアーキテクチャを保存するためのスライド調製方法

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

裏打ちクリア: 組織の脂肪組織の三次元イメージング法をクリア

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

4倍のチェッカーボード:薬物の組み合わせを研究するための三次元チェッカーボードの変形

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

インビトロ 血管疾患モデリングおよび薬物検査のためのマウス胚性幹細胞を用いた血管新生の三次元発芽アッセイ

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

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

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