Supported by National Institutes of Health (DK55679, and DK59888 to A.N.). Emory University&#160;Integrated Cellular Imaging Microscopy Core of the Winship Cancer&#160;Institute comprehensive cancer center grant, P30CA138292, and the Crohn's and Colitis Foundation of America Career Development award to C.T.C. and Fellowship Award to A.E.F.

実験は、年齢の10と12週間の間に、C57BL / 6マウスを使用していました。動物を用いたすべての手順を見直し、エモリー大学施設内動物管理使用委員会によって承認され、衛生基準の国立研究所に従って行ったしました。 1。実験のセットアップ<ol><li>クライオスタットの設定<ol><li>ミクロトームの刃とチャックを含む、-20℃にクライオスタットを平衡化（ブレード周り細心の注意を払って！）。 </li><li> 10月と空のcryomold（最適切断温度化合物）を記入し、-20℃（〜30分）に平衡化。金型から冷凍10月を外し、液体OCTでチャックに固定します。ミクロトーム腕でブロック/チャックを置き、10月は10分間に設定することができます。 </li><li>セクションごとに10μmのクライオスタットを設定し、それがフラットになるまで、10月ブロックを剃ります。数センチ離れブレードからミクロトーム腕をバックアップします。この時点で、EXPERの残りのブレードやチャックを調整していません形態。 </li></ol></li><li>かみそりの刃を準備します。サンプルあたり2かみそりの刃を準備します。金属ファイルを使用して、ブレードの尖鋭端鈍いです。次に、鉗子でアルミフランジを削除します。 </li><li>ドライアイス上でパイレックス皿などの平らな面を事前に冷やし。 </li><li> 10-15解剖ピンと解剖皿を準備します。シリコンが充填された10cmの組織培養皿50％は十分です。 RTでハンクス緩衝液5mlを追加します。 </li></ol>遠位結腸組織の2解剖<ol><li>密閉されたチャンバー内にマウスを置き、腹部と胸部12上に70％エタノールを噴霧し、その後、イソフルラン吸入と頸椎脱臼によりマウスを安楽死させます。 </li><li>はさみで腹部の皮膚の小さな切開を行い、その後、腹腔を露出させるために切開を行います。同様の手順は、12ここで説明されています。 </li><li>肛門縁で大腸を切り取り、コロンがフリーになるまで、腸間膜を離れていじめます。 </li><li>遠位結腸を切除肛門から0と6センチメートル間のセグメント。ここでは、陰窩の深さは〜150μmである（ 図1B参照 ）です。縦方向にはさみを使用して、コロンを開き、糞便内容を削除カット。 </li><li>粘膜表面を上に向けて解剖皿に組織をピン。ハンクス緩衝液中のシリコン皿上の組織使用して、解剖ピンを伸ばし、RT（ 図1C）で10分間休憩しましょう。これは、組織からひだを除去し、筋肉層がリラックスすることができます。 </li></ol>セクショニング用クライオスタット3.マウント組織<ol><li>組織の目的のセクションの下にカミソリの刃をスライドさせ、その後、ハンクス緩衝液を捨てます。サンドイッチを作り、トップに別のカミソリの刃を追加します。組織セグメントを切断し、ピンを取り外します。ドライアイスにカミソリの刃/組織サンドイッチを転送します。 （5〜10分間、 図1D）組織が ​​凍結することができます。 </li><li>カミソリ/組織サンドイッチをピックアップし、それがトップBLに指を置くことで少し温めますADE（粘膜側を上に。基底筋層が最初に区分されるように、これは、組織を配向します。）。サンドイッチを分離し、組織セグメントのエッジの周りカット。組織セグメント約0.5センチ×1センチカット。エッジ領域は連続切片は、多くの組織層を含有させ、カールする傾向を有します。オーバーカットアンダーカットよりも良好であることに注意してくださいので、十分過ぎるくらいの注意が必要。 </li><li>組織の上に氷が溶け始めるまで組織が加温することができます。この時点で、迅速かつ慎重にクライオスタット内のOCTブロックに組織を押してください。 </li><li>サンプルの上に指を置くことによって、ブレードを取り外します。熱伝達には、組織を破壊することなく、ブレードを削除することができます。 </li><li>コー​​ト10月で組織し、5分間平衡化。 10ミクロン（ 図1E、F）のセクションをカット。陰窩が見られるまで、視覚的にセクションを検査します。 1.5ミリリットルチューブ内またはスライド上のセクションを配置します。 </li><li> mRNAまたはタンパク質の分析のために、Tを配置5陰窩ベース、5移行、および管当たり5面部分とチューブで、彼のサンプル。 RNA回収のために1ミリリットルを商業分離試薬を追加し、室温で10分間インキュベートします。製造業者の説明書に従ってRNA精製を実行します。 cDNAの生成のための全RNAの5〜10μgのを使用してください。 </li><li>タンパク質精製のために、5つのセクションごとに寒RIPA溶解バッファー（プラスプロテアーゼ阻害剤）の0.5ミリリットルを追加します。氷上で、70％のサイクルで3回超音波処理。 <ol><li> SDS PAGEローディング緩衝液（Laemmli緩衝液）に試料を添加し、10分間100℃でインキュベートします。 SDS-PAGEの転送の対象サンプルは、その後、2時間の5％ミルク/ PBSでブロックします。 （1：500）、4℃でO / N KLF4抗体とのインキュベーション、続いてウエスタンブロット分析を実行します。 </li></ol></li></ol>

<ol>
	<li>Noah, T. K., Donahue, B., Shroyer, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+development+and+differentiation.">Intestinal development and differentiation.</a> Exp Cell Res. 317, 2702-2710 (2011).</li><li>Humphries, A., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+crypt+organization+and+tumorigenesis.">Colonic crypt organization and tumorigenesis.</a> Nat Rev Cancer. 8, 415-424 (2008).</li><li>Weber, C. R., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease:+is+it+really+just+another+break+in+the+wall.">Inflammatory bowel disease: is it really just another break in the wall.</a> Gut. 56, 6-8 (2007).</li><li>Bjerknes, M., Cheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+isolation+of+intact+epithelium+from+the+mouse+intestine.">Methods for the isolation of intact epithelium from the mouse intestine.</a> Anat Rec. 199, 565-574 (1981).</li><li>Sato, 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Rand, M. D., 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=Calcium+depletion+dissociates+and+activates+heterodimeric+notch+receptors.">Calcium depletion dissociates and activates heterodimeric notch receptors.</a> Mol Cell Biol. 20, 1825-1835 (2000).</li><li>Gjorevski, N., Boghaert, E., Nelson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Epithelial-Mesenchymal+Transition+by+Transmission+of+Mechanical+Stress+through+Epithelial+Tissues.">Regulation of Epithelial-Mesenchymal Transition by Transmission of Mechanical Stress through Epithelial Tissues.</a> Cancer Microenviron. 5, 29-38 (2012).</li><li>Lechner, 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=Gene+expression+pattern+of+laser+microdissected+colonic+crypts+of+adenomas+with+low+grade+dysplasia.">Gene expression pattern of laser microdissected colonic crypts of adenomas with low grade dysplasia.</a> Gut. 52, 1148-1153 (2003).</li><li>Reizel, 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=Colon+stem+cell+and+crypt+dynamics+exposed+by+cell+lineage+reconstruction.">Colon stem cell and crypt dynamics exposed by cell lineage reconstruction.</a> PLoS Genet. 7, e1002192 (2011).</li><li>Kosinski, C., 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=Gene+expression+patterns+of+human+colon+tops+and+basal+crypts+and+BMP+antagonists+as+intestinal+stem+cell+niche+factors.">Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors.</a> Proc Natl Acad Sci U S A. 104, 15418-15423 (2007).</li><li>Tamura, 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=Pit+pattern+and+three-dimensional+configuration+of+isolated+crypts+from+the+patients+with+colorectal+neoplasm.">Pit pattern and three-dimensional configuration of isolated crypts from the patients with colorectal neoplasm.</a> J Gastroenterol. 37, 798-806 (2002).</li><li>Geem, D., Medina-Contreras, O., Kim, W., Huang, C. S., Denning, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+dendritic+cells+and+macrophages+from+the+mouse+intestine.">Isolation and characterization of dendritic cells and macrophages from the mouse intestine.</a> Journal of visualized experiments : JoVE. , e4040 (2012).</li><li>Laukoetter, M. G., 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=JAM-A+regulates+permeability+and+inflammation+in+the+intestine+in+vivo.">JAM-A regulates permeability and inflammation in the intestine in vivo.</a> J Exp Med. 204, 3067-3076 (2007).</li><li>Neumann, P. 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=Gut+commensal+bacteria+and+regional+Wnt+gene+expression+in+the+proximal+versus+distal+colon.">Gut commensal bacteria and regional Wnt gene expression in the proximal versus distal colon.</a> Am J Pathol. 184, 592-599 (2014).</li><li>Capaldo, C. 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=Proinflammatory+cytokine-induced+tight+junction+remodeling+through+dynamic+self-assembly+of+claudins.">Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins.</a> Mol Biol Cell. 25, 2710-2719 (2014).</li><li>Wells, J. M., Spence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+an+intestine.">How to make an intestine.</a> Development. 141, 752-760 (2014).</li></ol>

The authors have nothing to disclose

結腸上皮細胞の増殖および分化に関与する分子メカニズムはほとんどわかっていません。これは、結腸上皮陰窩の基部に幹細胞区画の細胞シグナル環境の理解のギャップを含んでいます。腸細胞の分化の根底にあるプロセスが注目もあります。例えば、in vivoでの分化の理解がin vitroでの主な腸システム16を製造することを目的とした研究のためのベンチマークとして必要とされる増加しました。 上記の手順は、特別な注意を必要とするいくつかのステップが含まれています。まず、（セクション3.2で説明したように）凍結組織セグメントの周りのエッジを削除は解剖や凍結時のカール組織端として必要とされます。これらのエッジ結果を移動しないと、表面及び陰窩ベース細胞の混合集団です。予め冷却し、水平に10月ブロックの組織をマウントすることも不可欠です。 Tそのプロトコルは、各プール内のセクションの数を変更することによって遠位結腸の他の領域に適用することができます。 図1Bに示すように、例えば、粘膜陰窩の長さは150〜ミクロンの間で変化します。肛門4からCM-6センチメートルとの間の領域を研究する際に、セクションの数が重要な30に15から増加する必要があるため、このプロトコルは、（原因折り目に近接結腸（7センチメートル-9 cm）のために提案されていません襞obliquae）それは不可能連続切片によって表面及び陰窩ベース細胞を分離することルーメン内に延びています。 連続切片技術は陰窩ベースを研究し、ヒトにおける上皮細胞集団を表面に使用されてきました。しかし、我々のプロトコルが原因で、マウス組織のサイズが小さいために必要な追加のステップを追加します。我々は、上記のプロトコルは、遺伝的に扱いやすいマウスシステムにおける陰窩塩基および表面上皮細胞集団の調査を可能にすることを提案します。確かに、プールされたセクションでは、yieLD、高品質のタンパク質やRNAの適当な下流分析。将来のアプリケーションは、細胞シグナル伝達経路の研究やクロマチン免疫沈降を含むことができます。しかし、前述の代替の方法のいくつかのように、得られた切片は、上皮および間質固有層細胞の混合物を含むことに留意すべきです。結論として、ここで説明するプロトコルは、マウス結腸粘膜の空間的に異なる領域での分子プロセスの分析のための迅速で、高収率の方法を提供します。

結腸の上皮層は、常駐幹細胞は数日ごとに1組織の補充を有する高度に再生組織です。再生のこのプロセスは、増殖性の幹細胞区画の維持を必要とします。時間が経つにつれて、新たに生成娘細胞はその増殖能を失い、腸の管腔表面に向かって移動します。移行と一致し、前駆細胞は、成熟した障壁形成腸細胞や粘液産生杯細胞に分化します。この分化プログラムの障害は、炎症性腸疾患および大腸癌2,3に見られるような上皮バリアを妥協に寄与すると考えられています。 上記のプロセスの詳細な研究は、陰窩ベースと表面の細胞集団を単離するための方法論が必要になります。複数の方法は、独自の利点と欠点を有するそれぞれ、存在します。腸上皮細胞の単離のための現在の方法は、CHを含みます実験条件4,5に応じて全体の陰窩、上皮シート、または単一細胞の単離、その結果、薬剤および機械的解離をelating。これらは、高収量法である、まだ間シグナル伝達の変化は、ネイティブ環境6,7を表していない可能性がこれらの条件の下で報告されています。レーザーキャプチャーマイクロダイセクションは、空間的に異なる材料の収集を可能にしますが、低分子降伏8,9を実現しています。ヒト結腸組織では、連続水平凍結切片法10を開発してきました。しかし、マウスの粘膜組織は、この技術の直接処分のために法外小さいです。ヒトでは、腸の陰窩の長さ（陰窩ベースと表面の細胞間の距離）結腸では、〜100〜1000μmの11との間で変化します。マウスでは、陰窩の長さは、50〜300ミクロン〜の間（ 図1Aを参照）です。マウス陰窩のサイズが小さいイゾラへの挑戦を提示します既存のプロトコルを使用して、これら2つの細胞集団の化。 現在、陰窩ベースの単離のための低コスト、高歩留まり方法を説明し、マウスの結腸組織における上皮細胞集団を表面。このようにして採取した組織は、その後、免疫蛍光染色、RT-PCR（リアルタイムポリメラーゼ連鎖反応）、またはウェスタンブロッティングなどの標準的な下流のアプリケーションの数によって分析することができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems, Nussloch Germany</td>
			<td>CM 1510-3</td>
			<td></td>
		</tr>
		<tr>
			<td>MyiQ real time PCR detector</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 510&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek, Sakura Finetek, Torrance CA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>cryo-mold</td>
			<td>Tissue-Tek, Sakura Finetek, Torrance CA</td>
			<td>4557</td>
			<td>25mmx20mmx5mm</td>
		</tr>
		<tr>
			<td>High profile microtome blade</td>
			<td>Leica Biosystems, Nussloch germany</td>
			<td>818</td>
			<td></td>
		</tr>
		<tr>
			<td>GEM industrial blades</td>
			<td>American Safety Razor Blades</td>
			<td>62-0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard silicon elastomere base</td>
			<td>Dow Corning, Midland MI</td>
			<td>3097358</td>
			<td></td>
		</tr>
		<tr>
			<td>27 1/2 g needle</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>305109</td>
			<td>pins for dissection</td>
		</tr>
		<tr>
			<td>TRizol</td>
			<td>Life Technologies</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody ZO-1</td>
			<td>Invitrogen&#160;</td>
			<td>187430</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody KLF4</td>
			<td>Cell Signaling</td>
			<td>4038S</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody mGAPDH</td>
			<td>Sigma</td>
			<td>G8795</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody Secondary Alexa conjugate</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>488 anti-rabbit</td>
		</tr>
		<tr>
			<td>Antibody Secondary HRP conjugate</td>
			<td>Jackson</td>
			<td>111/115-001-003</td>
			<td>rabbit/mouse&#160;</td>
		</tr>
		<tr>
			<td>Topro-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>HANKs balanced salt buffer</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td></td>
			<td></td>
			<td>50mM Tris-HCl pH 7.4 150mM NaCl 1% NP40 0.25% Na-deoxycholate</td>
		</tr>
		<tr>
			<td>PCR Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>primer TBP 3&#39;</td>
			<td></td>
			<td></td>
			<td>GGAATTGTACCGCAGCTTCAAA</td>
		</tr>
		<tr>
			<td>primer TBP 5&#39;</td>
			<td></td>
			<td></td>
			<td>GATGACTGCAGCAAATCGCTT</td>
		</tr>
		<tr>
			<td>primer KLF4 3&#39;</td>
			<td></td>
			<td></td>
			<td>TGTGACTATGCAGGCTGTGGCAAA</td>
		</tr>
		<tr>
			<td>primer KLF4 5&#39;</td>
			<td></td>
			<td></td>
			<td>ACAGTGGTAAGGTTTCTCGCCTGT</td>
		</tr>
	</tbody>
</table>

cryosectioning method for microdissection of murine colonic mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

結腸組織切片を組織学的評価およびH＆E染色のために処理した13。粘膜の伝統的な断面図を図2Aに見られます。基礎分野は、主に円形の筋層から成る外筋層が含まれています。内腔に向かって進むと、粘膜筋板は、陰窩 - ベースの上皮細胞に隣接して明らかである、移行細胞は、組織の真ん中にあり、最終的に、表面細胞集団は、腸管内腔に面し。ここで説明した連続切片法は粘膜筋板（ 図2C）、続いて縦方向および円形の筋層が最初に遭遇するような姿勢（ 図2B）を 、保持しています。画像の左上にある非筋間質細胞の出現に注意してください。以下に説明する下流の分析のため、筋切片は廃棄されます。以降のセクションでは、上皮細胞と間質組織（固有層を含んでいます）、陰窩ベース細胞（ 図2D）で始まります。による密に充填された核に暗く見える陰窩のほとんどにおいて中央管腔が存在しないことに注意してください。これらの層は、増殖コンパートメントが含まれています。移行細胞は顕著な中央ルーメン（ 図2E）と密に詰まった腺として表示されます。最後に、表面の細胞集団は、 顔 （ 図2F）JP見上皮単層の領域と、不規則な構造を有しています。 （ここでは14のように）上記のように連続切片はまた、免疫蛍光標識および共焦点顕微鏡法のために処理することができる。 図2Gは 、核（青）および細胞-細胞接合タンパク質閉鎖帯について染色し、続いて固定し、100％メタノールで透過処理し、単離された陰窩を示しOccludens 1（ZO-1、緑）。伝統的な切片の向きでは、cryptと表面の細胞を同時に表示することができます（ 図2G </stronG&gt;）。結腸陰窩の内腔ZO-1接合部の汚れラインことに注意してください。管腔のこのZ0-1裏地も円形腺（ 図2G）の中央に、移行細胞集団の連続切片試料に見られます。拡張ZO-1染色は表面細胞集団（ 図2IおよびJ）に見られます。 いくつかの分化因子は、陰窩ツー面軸に沿って時空間的様式で発現されることが知られています。これは、表面の細胞集団が富化されることが知られている転写因子クルッペル様因子4（KLF4）を含みます。従来の切片法を用いて、KLF4、表面細胞集団（ 図2K）に対向管腔の核内に見出されます。そこで、KLF4 mRNAは主に表面細胞集団内で発現されるであろうと推測しました。これをテストするには、連続切片を採取し、表面、移行、および陰窩ベースサンプルに分類しました。 Subsequently、RNAは、RNeasyカラムとDNase処理により、さらに精製して、標準のTrizol抽出を用いて回収しました。 RNAは、サンプル当たりの総RNAの5-10μgの間に得られたプールされた5個の連続切片から回収しました。これらのサンプルは、その後、KLF4および参照遺伝子、TATAボックス結合タンパク質1（TBP-1）（ 図2L）14の両方に、半定量的リアルタイムPCRを行いました。 図2Lに示すように、TBP-1に正規化した後に、表面細胞は陰窩細胞中で発現される8回KLF4 mRNAにまで発現することが見出されました。同様に、KLF4タンパク質レベルは、陰窩面軸に沿って空間的調節を示すことが見出されました。上述した後、4℃で20分間、溶解緩衝液中でインキュベートしたように連続切片をプールしました。これは、サンプルあたりのタンパク質の200〜250μgの間に生じました。次に、試料をSDS-PAGE（ 図2M）15による分析を行いました。 図2Mに示すように、KLF4のProteinレベルは劇的に表面細胞集団において高いです。上記の知見は、マウス結腸粘膜から表面及び陰窩の上皮細胞を大量に単離するためのこのプロトコルの能力を実証します。 <img alt="図1" src="/files/ftp_upload/53112/53112fig1.jpg" /> 図1：マウス結腸粘膜および外部筋肉層を示す（A）の回路図。我々の方法は、シリアル凍結切片（10μmの各）により離散表面細胞および陰窩ベースの細胞集団を収集することを目的とします。外部筋肉層は破棄されます。 （B）墓所の高さは、コロン（陰窩ベースと表面層との間の距離）の長さに沿って変化します。データポイントは、個々の陰窩測定および記号は生物学的複製を示す指示（N = 4）。 （C）結腸セグメントは、収集二分、およびシリコン解剖皿に固定します。 SE（D）組織肛門からgment約3cmはカミソリの刃の間でプレスし、ドライアイスで凍結されています。 （E）組織は、その後、平坦化前のOCTブロックに取り付けられています。 （F）凍結切片を取り出し、目視で検査します。 <img alt="図2" src="/files/ftp_upload/53112/53112fig2.jpg" /> 図2：代表的なデータの円形筋（CM）を示す断面図でヘマトキシリン-エオシンで染色した（A）結腸組織、粘膜筋板（mm）で、陰窩-基本セル、移行細胞、および表面の細胞。 （B - C）結腸筋層。 （D）クリプト系細胞。中央管腔が存在しないことに注意してください。 （E）移行細胞。 （F）表面細胞。単離された結腸陰窩の（G）免疫蛍光染色。 ZO-1（緑）および核（青）は、断面で観察されます。 （HI）ZO-1と移行し、表面の細胞で核染色。 （J）ZO-1染色の拡大図。 （K）、KLF4（緑）は、表面の細胞集団において濃縮されます。 （L）3つの区画の間にKLF4 mRNAレベルのリアルタイムPCR評価。 （M）は、これらの細胞集団におけるKLF4タンパク質レベルのウェスタンブロット分析。

Watch this Scientific Journal Video about Cryosectioning Method for Microdissection of Murine Colonic Mucosa at JoVE.com

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

マウス結腸粘膜の顕微解剖のための凍結切片法

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

cryosectioning-method-for-microdissection-of-murine-colonic-mucosa

Research

JoVE Journal

Biology

17.2K ビュー.  Emory University, Atlanta, Georgia. Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

ビデオ: Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Flashは、E12.5マウス脳の凍結とCryosectioning

生物学

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

哺乳動物の組織のレーザーキャプチャーマイクロダイセクション

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

ゼブラフィッシュ胚眼の組織の顕微解剖

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

ブラックウィドウスパイダーシルクを生産腺の顕微解剖

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

人間の腸粘膜の外植片の文化と偏刺激する新規な方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

蛍光パターンを調査するための凍結セクショニング酵母コミュニティ

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

個々の細胞タイプの転写プロファイリングのためのツールと​​してのレーザ支援顕微解剖（LAM）

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

遺伝子発現と組織学的分析のためにシングルマウス骨格筋の連続領域の凍結切片化

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

形態とマウスの下顎頭軟骨細胞解析法

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

遺伝子発現解析のためのマウスの目から非常に純粋な小柱網のキャプチャ マイクロダイゼクション

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...