私がリー研究所にいたとき、このプロジェクトを監督してくれたジニー・T・リーと、原稿を編集してくれたタイラー・ブローリングに感謝します。この作品は、ダイムズ財団のマーチからバジル・オコナースタータースカラー賞とNIHグラントGM098605によって支えられました。

1.3D スライドの準備
<ol><li>以下の試薬を準備します。<ol>
<li>CSKバッファ:100 mM NaCl、300 mMスクロース、10 mMパイプ、3 mM MgCl2.1 M NaOH で pH を 6.8 に調整します。CSKバッファーは4°Cで保管してください。</li>
		<li>0.5%トリトンX-100のCSKバッファ:200 μlのトリトンX-100から40 mlのCSKバッファを加えます。トリトンX-100が完全に溶解するまで、磁気撹拌機を使用して溶液を混合します。CSKバッファーを0.5%トリトンX-100で4°Cで保管してください。</li>
		<li>4% パラホルムアルデヒド (PFA)-PBS、 pH 7.4: 約 70-80 ml の水に PFA の 4 g を溶解します。溶解プロセスを迅速化するために、約20 μlの4 N NaOHを加え、PFAが完全に溶解して溶液が透明になるまで、水浴中の溶液を60°Cに保ちます。このプロセスは約1時間かかります。PFAが完全に溶解した後、10x PBSの10mlを加えます。水で最終体積を100mlに調整し、溶液を室温(25°C)に冷却します。新しい実験ごとに、新たに準備したソリューションを使用します。 注:すべての動物の仕事のための制度的許可を得て、関連する動物のケアガイドラインに従ってください。</li>
	</ol>
</li>
	<li>雄マウスを安楽死させる。鉗子とはさみを使用して、マウスから精巣を切除する。テストをPBS(またはRPMI 1640)に入れる。精巣を除去した後、RNAの分解を防ぐために、以下のステップ1.3-1.4を迅速に実施する。</li>
	<li>小さなペトリ皿の精巣を破裂させ、 チュニカアルブギネアを取り除く.鉗子を使用して氷の上のPBSで半日本の細管を解き明かす。</li>
	<li>鉗子を使用して、500 mlのCSKバッファー+0.5%トリトンX-100を含む4ウェル皿の1つの井戸に、いくつかの管状(長さ約5〜10mmの細管)を1つの井戸に移し、6分間インキュベートする。CSKバッファーへの過度の露出は、細胞の破壊につながる可能性があります。</li>
	<li>鉗子を使用して、4%PFA-PBS溶液を含む4ウェル皿の1つの井戸にすべての細管を移す。このステップは、実体顕微鏡なしで前形をすることができるが、任意で使用することができる。室温で10分間インキュベートします。同時にインキュベーション期間中に12個の細胞スピンチャンバーの準備を開始する。</li>
	<li>鉗子を使用して、すべての細管をPBSを含む4ウェル皿の1つの井戸に移します。室温で5分間インキュベートします。</li>
	<li>ガラススライドの裏側で、すべての管を30 μlのPBSに移します(カチオン電荷のため、スライドの上部は避けてください)。2つの鉗子の先端を使用して、細管を粉々に引き裂く。2つの鉗子の先端の間で管を切り取り、鉗子を水平に引っ張ることによって、管を水平方向に刻みます。この手順を約 10 ~ 20 秒間続けます。</li>
	<li>P20ピペットを用いてピペットで懸濁液を混ぜます。</li>
	<li>懸濁液をマイクロ遠心チューブに移し、PBSで約1.3mlに希釈します。12個のサイトスピンチャンバ(合計1.2 ml)のそれぞれに100 μlの懸濁液を塗布します。この工程では、細胞の濃度を決定する必要はありません。</li>
	<li>細胞回転は、室温で10分間2,000rpmでサンプルを回転させます。サイトスピニング後、実験室のベンチで室温で数分間スライドします。スライドが数分完全に乾燥した後、次のステップに進みます。</li>
	<li>PBSを含むコプリン瓶を使用して、室温で5分間スライドを洗浄します。</li>
	<li>70%エタノールを含むコプリン瓶にスライドを移し、RNA FISHの開始前に少なくとも2分待ちます。スライドは、4°Cで70%エタノール中の数週間安定である。 長期保存の場合、コプリン瓶に80%と100%エタノールを使用して2分間連続処理を施し、スライドを完全に脱水乾燥します。スライドボックスに-80°Cで保管してください。</li>
</ol>2. ランダムプライミングによるコト-1 DNAプローブ調製
<ol><li>以下の試薬を準備します。<ol>
<li>ハイブリダイゼーションバッファ:ハイブリダイゼーションバッファストックの900 mlを準備するために進行コンポーネントを混合します。このソリューションは、プローブのストックに使用されます。このハイブリダイゼーションバッファーストックは、ハイブリダイゼーションの前にRNase阻害剤溶液(リボヌクレオシドバナジル錯体)の10%の体積の添加を可能にします。<table><tbody>
<tr>
<td>コンポーネント</td>
					<td>量(μl)</td>
					<td>最終濃度</td>
				</tr>
<tr>
<td>ホルムアミド</td>
					<td>500</td>
					<td>50% (v/v)</td>
				</tr>
<tr>
<td>デキストラン 50%</td>
					<td>200</td>
					<td>10% (w/v)</td>
				</tr>
<tr>
<td>20x SSC</td>
					<td>100</td>
					<td>2x</td>
				</tr>
<tr>
<td>1% BSA</td>
					<td>100</td>
					<td>0.1%</td>
				</tr>
</tbody></table>ハイブリダイゼーションの前に、200 mM リボヌクレオシド バナジルコンプレックスまたは水の 1/10 vol を最終濃度に合わせてハイブリダイゼーション バッファー ストックに加えます。-20°Cでは、この混合物は1年以上安定している。</li>
		<li>20x SSC:クエン酸ナトリウム88.2g、NaCl175.3gを約800mlの水に溶かします。1 M HCl の数滴を使用して pH を 7.0 に調整し、水を加え、オートクレーブ処理で最終ボリュームを 1 L. 滅菌に調整します。調製後、20倍のSSCを室温で保存することができます。</li>
		<li>50%(w/v)デキストラン:水をかき混ぜ、ゆっくりと50gのデキストラン硫酸塩を加えます。室温で一晩かき混ぜ続けます。水を加えると、最終容積を100mlに調整します。室温で保管してください。</li>
	</ol>
</li>
	<li>以下の成分を PCR チューブに追加します。PCR機を使用して、95°Cで5分間混合してインキュベートします。<table><tbody>
<tr>
<td>コンポーネント</td>
				<td align="center">反応あたりの量 (μl)</td>
				<td align="center">最終的な</td>
			</tr>
<tr>
<td>コト-1 DNA:1 mg/ml</td>
				<td align="center">1</td>
				<td align="center">1 μg</td>
			</tr>
<tr>
<td>ランダム9マープライマー(キットから)</td>
				<td align="center">10</td>
				<td align="center"></td>
			</tr>
<tr>
<td>水</td>
				<td align="center">27</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>遠心分離機は簡単に氷の上に置きます。</li>
	<li>次のコンポーネントを追加します。PCR機を使用して37°Cで30分間混合し、インキュベートします。(使用したキットの詳細については、試薬および装置の表を参照してください。<table><tbody>
<tr>
<td>コンポーネント</td>
				<td align="center">反応あたりの量 (μl)</td>
				<td align="center">最終濃度</td>
			</tr>
<tr>
<td>ステップ2.2から調製された混合物</td>
				<td align="center">38</td>
				<td align="center"></td>
			</tr>
<tr>
<td>5xヌクレオチドバッファー(キットから)</td>
				<td align="center">9.2</td>
				<td align="center"></td>
			</tr>
<tr>
<td>サイ3-dUTP (1 mM)</td>
				<td align="center">0.8</td>
				<td align="center">16 μM</td>
			</tr>
<tr>
<td>クレノウ (キットより)</td>
				<td align="center">2</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>2 μlのストップバッファー(キットから)を加えると、反応を中止します。</li>
	<li>マイクロスピンカラムを使用して停止した反応を精製します。</li>
	<li>精製された分率に50μg(5μl)のニシン精子DNA(鋳型Cot-1 DNAの50倍の過剰量)を加えます。</li>
	<li>ピペットを使用して総体積を測定します。0.7倍のエタノール量100%、酢酸ナトリウム3M(pH 5.2)の0.1倍の体積を加え、よく混ぜ、遠心分離機を最大速度(17,000 x g)で4°Cのマイクロ遠心分離機を使用して15分間加えます。</li>
	<li>液体を取り出し、200μlの70%エタノールを加え、4°Cで最高速度で5分間よく混ぜます。</li>
	<li>余分な液体を取り除きます。沈殿物を室温で10〜30分間乾燥させます。</li>
	<li>20 μlのハイブリダイゼーションバッファーストックにペレットを溶かします。ペレットは溶解しにくいことが多いため、ピペットを繰り返し使用する。代替オプションとして、ボルテックスミキサーを使用してペレットを効率的に溶解することもできます。プローブは使用するまで-20°Cに保つことができます。</li>
</ol>3. コト-1 RNAフィッシュ
<ol><li>以下のコンポーネントを PCR チューブに追加します。PCR機を用いて、80°Cで10分間インキュベートし、続いて42°Cで約10〜30分間の前焼成工程を行う。 ハイブリダイゼーションが行なくなるまで42°Cに保ちます。リボヌクレオシドバナジル複合体は任意です。それは水と交換することができます。<table><tbody>
<tr>
<td>コンポーネント</td>
				<td align="center">反応あたりの量 (μl)</td>
				<td align="center">最終的な</td>
			</tr>
<tr>
<td>コト-1 DNAプローブ(50 ng/μl)</td>
				<td align="center">2</td>
				<td align="center">100 ng</td>
			</tr>
<tr>
<td>リボヌクレオシド・ヴァナジル・コンプレックス (200 mM)</td>
				<td align="center">2</td>
				<td align="center">20mM</td>
			</tr>
<tr>
<td>ハイブリダイゼーションバッファ</td>
				<td align="center">20 に追加</td>
				<td align="center"></td>
			</tr>
</tbody></table>
</li>
	<li>80%エタノールを含むコプリン瓶で2分間脱水スライドし、続いて100%エタノールを含むコプリン瓶を2分間脱水する。室温の実験室のベンチの乾燥スライドは完全に。</li>
	<li>42°Cインキュベーターにチップボックスチャンバー(2〜3cmの水で満たされた)を予熱します。先端箱の部屋の上にスライドを置きます。プレアンネプローブとピペットを脱水スライドに直接遠心分離します。泡を作らないようにしてください。スライドをカバースリップでそっと覆います。</li>
	<li>42°Cで6時間から一晩(14〜15時間)ハイブリダイズする。</li>
	<li>コプリンの瓶(2x SSCと50%のフォルミドを備えた2つの瓶、2倍のSSCを備えた2つの瓶)で洗浄液を準備します。42°Cで水浴で30分間予熱します。</li>
	<li>かみそりの刃を使用して、スライドの端からカバースリップを取り外します。サンプルを引っ掻かないようにします。2x SSCと50%ホルムアミドを含むコプリン瓶にスライドを45°Cで5分間洗浄します。 この手順を 1 回繰り返します。</li>
	<li>2x SSCを含むコプリン瓶にスライドを5分間45°Cで洗います。 この手順を 1 回繰り返します。</li>
	<li>スライドに20μlのDAPI取り付けメディアを追加し、カバースリップでカバーします。カバーガラスを軽く押して、余分な取り付けメディアを取り外します。洗浄ティッシュと余分な土台媒体をブロット。スライドは顕微鏡評価の準備ができています。</li>
</ol>

<ol>
	<li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Namekawa, S. 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=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr Biol. 16, 660-667 (2006).</li><li>Namekawa, S. H., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+nascent+RNA,+single-copy+DNA+and+protein+localization+by+immunoFISH+in+mouse+germ+cells+and+preimplantation+embryos.">Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos.</a> Nat Protoc. 6, 270-284 (2011).</li><li>Mahadevaiah, S. K., Costa, Y., Turner, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+RNA+FISH+to+study+gene+expression+during+mammalian+meiosis.">Using RNA FISH to study gene expression during mammalian meiosis.</a> Methods in molecular biology. 558, 433-444 (2009).</li><li>Hall, L. L., 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=An+ectopic+human+XIST+gene+can+induce+chromosome+inactivation+in+postdifferentiation+human+HT-1080+cells.">An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.</a> Proc Natl Acad Sci U S A. 99, 8677-8682 (2002).</li><li>Huynh, K. D., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+a+pre-inactivated+paternal+X+chromosome+in+early+mouse+embryos.">Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos.</a> Nature. 426, 857-862 (2003).</li><li>Namekawa, S. H., VandeBerg, J. L., McCarrey, J. R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+chromosome+silencing+in+the+marsupial+male+germ+line.">Sex chromosome silencing in the marsupial male germ line.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9730-9735 (2007).</li><li>Namekawa, S. H., Payer, B., Huynh, K. D., Jaenisch, R., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+imprinted+X+inactivation:+repeat+versus+genic+silencing+in+the+mouse.">Two-step imprinted X inactivation: repeat versus genic silencing in the mouse.</a> Mol Cell Biol. 30, 3187-3205 (2010).</li><li>Ichijima, Y., 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=MDC1+directs+chromosome-wide+silencing+of+the+sex+chromosomes+in+male+germ+cells.">MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.</a> Genes Dev. 25, 959-971 (2011).</li><li>Sin, H. S., 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=RNF8+regulates+active+epigenetic+modifications+and+escape+gene+activation+from+inactive+sex+chromosomes+in+post-meiotic+spermatids.">RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids.</a> Genes Dev. 26, 2737-2748 (2012).</li></ol>

著者は開示するものは何もない。

3Dスライドの準備では、パーメアビライゼーションステップは固定ステップの前に行います。これらのステップは、半細管上で直接行われ、それによって、3次元クロマチン構造を維持する。RNA/DNA FISHのクロマチン構造を保存するための代替オプションは、パーメアビライゼーションステップと固定ステップを同時に行うことである。この代替技術は、有袋類生殖細胞およびマウスの着床前胚<...

精巣では、生殖細胞は、二数葉化精子腫から、成熟したハプロイド精子に対するマイオシスを介して分化する。この過程において、胚芽細胞の核クロマチン構造は、連続的かつ動的に再モデル化される。表面の広がりは、個々の精子細胞の細胞学的検査に一般的に使用される。表面広がりの一般的な方法は、個々の精子細胞が広がり、平坦化される低調薬治療を採用する1.これらの条件は、精密染色体の詳細な分析に最適です。シナプス状態や再結合病巣などの染色体特徴は、この方法を用いて容易に観察される。しかし、低張治療は亜核クロマチンアーキテクチャを破壊し、したがってこの技術は核の構造解析には適さない。その結果、精巣生殖細胞の三次元クロマチン構造を維持するように改良された方法が設計されている。この方法は、3次元(3D)スライド法と呼ばれる(図1)。3Dスライド法は、その現場でのRNA蛍光による精巣胚芽細胞分化中の遺伝子発現およびクロマチン状態を調べるために最初に最適化されたため、核内の新生RNA局在化の検出を可能にした(FISH)2,3。この3D法は、免疫蛍光、DNA、RNA FISHの組み合わせにも適用可能です。さらに、この方法は、ポストメオティック性クロマチン(PMSC)の発見に役立ち、ポストメオティック精子2に見られる性染色体の無声コンパートメントである。
3Dスライド法は、核染色に一般的に使用されるスライド調製の2つの重要なステップの組み合わせによって、もともと最適化されました:核物質を固定するために設計された固定ステップと、抗体やFISHプローブなどの染色試薬の入手可能性を向上させるために細胞質材料を除去することを意図した透過性ステップ。他の刊行3で既に説明したように、低細胞質バックグラウンドで最適な結果を得るためには、パーメアビレーションステップが固定工程の前になければならないことが決定された。この3Dスライド法では、透過化ステップとその後の固定ステップを半細管上で直接行い、続いて細胞回転前に細胞を鉗子で生殖細胞を滑らかに回転させます。精子細胞のRNA FISHの代替方法が別の実験室4で開発された。この方法では、3D法と一致し、RNA FISHの最適な結果を得るためにパーメアビライゼーションステップが固定工程の前になければなりません。
以下のプロトコルは、3Dスライド法について説明し、核新生RNAの検出に可能な応用例であるCot-1 RNA FISHを提供する。Cot-1 DNAは、ゲノム中の反復的な要素から構成されています。ここで、Cot-1 DNAプローブは、新生転写物のイントロンとUTRにハイブリダイズされ、核5,6内の転写活性領域を検出する。3Dスライドは汎用性が高く、クロマチンアーキテクチャ間の空間的関係の詳細な検査を得るために、免疫蛍光、DNAおよびRNA FISH技術の組み合わせに適用することができます。

<table border="0" cellpadding="0" cellspacing="0" width="562">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cot-1 DNA&#160;</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stratagene Prime-It Fluor Fluorescence Labeling Kit</td>
			<td style="width:147px;">Agilent</td>
			<td align="right" style="width:119px;">
			300380
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herring sperm DNA Solution</td>
			<td style="width:147px;">Invitrogen</td>
			<td>15634-017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol, 200 proof (absolute)</td>
			<td>Pharmco-AAPER</td>
			<td align="right">111000200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Formamide deionized</td>
			<td>American Bioanalytical</td>
			<td>AB00600-00500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate tribasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">C8532</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Bovine serum albumin</td>
			<td style="width:147px;">Lifeblood Medical</td>
			<td style="width:119px;">77110-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulfate sodium salt from Leuconostoc spp.</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D6001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">A10491-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P6757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">M0250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">76240</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right" style="width:119px;">221465</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">P4417</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASHIELD Mounting Medium with DAPI</td>
			<td style="width:147px;">Vector Laboratories</td>
			<td style="width:119px;">H1200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonucleoside-vanadyl complex</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">S1402S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illustra MicroSpin G-50 Columns</td>
			<td>GE Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge</td>
			<td style="width:147px;">Eppendorf</td>
			<td align="right" style="width:119px;">5424</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Forceps</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">300-050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Microcentrifuge tubes</td>
			<td style="width:147px;">Bioexpress</td>
			<td style="width:119px;">C-3262-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cytospin 3</td>
			<td style="width:147px;">Shandon</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Coplin jar</td>
			<td style="width:147px;">Fisher</td>
			<td style="width:119px;">08-815</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dish</td>
			<td>Fisher</td>
			<td style="width:119px;">12-566-301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass&#160;</td>
			<td>Fisher</td>
			<td style="width:119px;">12-544-G</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Air incubator</td>
			<td style="width:147px;">Revolutionary Science</td>
			<td style="width:119px;">RS-IF-203</td>
		</tr>
	</tbody>
</table>

slide preparation method to preserve three-dimensional chromatin architecture of testicular germ cells

精巣胚芽細胞分化の間、核クロマチンの構造は動的に変化する。以下は、マウスに見られる精巣生殖細胞の三次元クロマチン配置を維持するために設計された方法を説明します;この方法は三次元(3D)スライド法と呼ばれる。この方法では、精巣細管は細胞質材料を除去する透過性のステップで直接処理され、続いて核物質を固定する固定ステップが続く。次いで、尿細管を解合し、細胞懸濁液を細胞紡ぎ、細胞はスライドに付着させる。この方法は、亜核組織の検出に対する感度を向上させ、免疫蛍光、DNA、および蛍光をその 中で の蛍光、DNA、およびRNA蛍光(FISH)およびこれらの検出方法の組み合わせに適用可能である。3Dスライド法の応用例として、Cot-1 RNA FISHが新生RNAを検出することが示されています。3Dスライド法は、精巣胚芽細胞分化中のクロマチン構造、DNA、RNA間の空間的関係の詳細な検討を容易にする。

3D スライドの代表的な結果を図 2に示します。この実験では、コト1RNA FISHを、抗CBX1およびγH2AX抗体を用いた免疫染色と共に新生転写を検出するために行った。RNA FISHと免疫染色を組み合わせるために、免疫染色を最初に行い、続いて3に記載されるRNA FISHを行った。CBX1は、XY体(または性体)と呼ばれるメイオシスにおける中心血球ヘテロクロマチンおよびサイレントセック?...

Watch this Scientific Journal Video about Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells at JoVE.com

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

ここでの材料は精巣生殖細胞の三次元クロマチン構造を維持するために開発された方法を記述する。これは三次元(3D)スライド法と呼ばれます。この方法は、亜核組織の検出感度を向上させ、免疫蛍光、DNA、および蛍光をその 中で の蛍光(FISH)に適用可能です。

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 ...

slide-preparation-method-to-preserve-three-dimensional-chromatin

Research

JoVE Journal

Biology

7.3K ビュー.  Cincinnati Children's Hospital Medical Center. ここでの材料は精巣生殖細胞の三次元クロマチン構造を維持するために開発された方法を記述する。これは三次元(3D)スライド法と呼ばれます。この方法は、亜核組織の検出感度を向上させ、免疫蛍光、DNA、および蛍光をその 中で の蛍光(FISH)に適用可能です。

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

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

三次元細胞培養用チップサイズの足場の微細加工

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

生物学

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, ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

三次元混合細胞スフェロイド共培養から乳腺上皮細胞の分離

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

精巣細胞懸濁液の調製のためのシンプルで効率的な手法

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

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 ...

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

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 system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

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 ...

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 ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

インタクトオルガノイドの単一細胞分解能3次元イメージング

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

3次元共培養システムNICO-1を用いた卵巣癌幹細胞様細胞の血管遺伝学的特性の評価

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...