Name: improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses
Uploaded: 2016-07-13T14:00:00
Description: Watch this Scientific Journal Video about Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses at JoVE.com

We would like to thank Ainara Ruiz de Sabando for providing H&amp;E images. This work was supported by grants from the National Institutes of Health (DK052230, DK093680 and CA172113) awarded to Dr. Vincent W. Yang.

1.マウス<ol><li>マウスを含むすべての研究は、ニューヨーク州立大学ストーニーブルック校施設内動物管理使用委員会（IACUC）によって承認されました。マウスは、12：12時間の明暗サイクルで維持しました。 <ol><li>商業的にC57BL / 6マウスを得ます。 loxP部位（L / F リットル F KLF5）によって隣接KLF5対立遺伝子を運ぶC57BL / 6マウスを入手し?...

<ol>
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スイスの圧延技術は​​、大規模で組織学的および形態学的評価のための腸組織を調製するための強力な方法です。もともと凍結切片18,19の製造のために開発された、以前に記載スイス圧延技術とは対照的に、ここで紹介する手順は、ホルマリン固定およびパラフィン包埋（FFPE）のプロンプト腸組織の準備と固定を可能にします。凍結組織と比較して、FFPE組織は、はるかに長い貯蔵?...

哺乳類の腸上皮は円柱細胞の単層を含みます。分化した細胞は、絨毛の領域を占有しながら、小腸において、増殖細胞は陰窩に閉じ込められます。大腸には​​絨毛がないのでしかし、増殖性細胞は陰窩の底に局在し、分化した細胞は陰窩の上部領域を占有しています。陰窩内の増殖細胞の連続した​​分割によって駆動される - （5日、約3）腸上皮は、迅速な補給を受けます。陰窩の増殖細胞は均質な集団ではなく、さらに、幹細胞とトランジット増幅（TA）のセル1に細分化されています。 2一番下から5細胞-幹細胞は、最初の4以内、陰窩の底部に存在します。陰窩ベース柱状（CBC）、幹細胞と予備静止幹細胞：現在のモデルは、2つの幹細胞の種類の存在をサポートしています。 CBCのTEM細胞が活発に増殖しているとロイシンリッチリピート含有Gタンパク質共役受容体5（LGR5）3、Olfactomedin 4（Olfm4）4とAchaeteの盾板状の2（Ascl2）5でマークされています。一方、予備静止幹細胞は、B細胞特異的モロニーマウス白血病ウイルス統合部位1（BMI1）6、マウステロメラーゼ逆転写酵素（mTert）7により標識され、HOPホメオボックス（Hopx）8、ダブルコルチン様およびCAMキナーゼ様1（DCLK1）9、およびロイシンリッチリピートおよび免疫グロブリン様ドメイン1（Lrig1）10。活発に増殖している幹細胞は、次いで、吸収細胞（腸細胞）および分泌細胞（腸、杯、パネート、およびタフト細胞）へのさらなる分化を受けるTA細胞を生じます。彼らはアポトーシスを起こすとされている絨毛のトップに到達するまで増殖帯での連続的な細胞分裂は、陰窩 - 絨毛軸に沿った上皮細胞の上方への移動をもたらします上皮の表面から脱落。腸上皮細胞の異なる種類は、異なるタンパク質（ 例えば 、腸杯細胞がリゾチームに対する抗体でMUC2及びパネート細胞に対する抗体で染色することにより認識することができる）の発現によって示されています。私たちは、腸上皮11-13の恒常性と病理生物学におけるクルッペル様因子（KLFs）の役割を研究します。変形スイス圧延技術の実現可能性を支持し、ここで示された結果は、活発に増殖し、腸上皮幹細胞14の維持にクルッペル様因子5（KLF5）の役割の以前の研究に基づいています。 KLF5は、高活性腸管幹及びTAのセル12内で発現されるジンクフィンガー転写因子です。以前の研究では、KLF5はのKi-67、腸陰窩で知られている増殖マーカーと共発現していることを実証しました。 胃腸のtr<html>行為は、構造的または機能的に均質な組織ではありません。小腸はさらに、近位、中間、および遠位の部分に分け、後者で、十二指腸、空腸、および回腸と盲腸および結腸に大腸に分割されています。これらの各セクションは、固有の組織学的特徴を有しており、別個の役割15を担っています。このように、傷害及び腸上皮の応答の程度の影響を研究した組織16の領域に依存してもよいです。さらに、種々のマウス系統は、試験16に使用される損傷の種類に基づいて、組織学的レベルでの応答の多様性を示します。これにより、組織標本にふさわしい、適切な組織学的および腸組織の分子の分析を可能にするために必要です。このように、スイスロール技術は一度に腸上皮の完全な長さの分析を許可するため、総合的な情報に基づいて、十分な情報に結論を確認します。 e_content &quot;&gt;スイスロールの技術は最初マグナス17言及し、MoolenbeckとRuitenbergパークらによって詳細に説明した。組織を調製し、組織を実行するための方法は、それぞれ、げっ歯類腸18,19を分析する。プロトコル診断目的のために、タイムリーかつ信頼性の高い組織調製のために許可する独自の方法の改良版は、この公報に提示描写。この修正された技術は、効率的なコレクションと、そのような免疫組織化学、免疫蛍光、ならびに普遍的に使用される技術のための腸管上皮の調製を可能にしますin situハイブリダイゼーション（蛍光及び発色20） のように。また、変更された組織標本の作製方法は、容易に入手可能で利用し、比較的安価な試薬の迅速な組織固定の方法を提供しつつ、さらなる評価のために、タンパク質、DNA、及びRNAの回収を可能にする。撮影します一緒に、THIsの技術は、腸上皮の、組織病理学的病理学、および分子的特徴を総合的に評価するための優れたです。

<table border="0" cellpadding="0" cellspacing="0" width="1016">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Stainless Steel Dissecting Kits</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">25640-002</td>
			<td style="width:403px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Decloaking Chamber</td>
			<td style="width:179px;">Biocare Medical</td>
			<td>DC2012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Syringe 10ml</td>
			<td style="width:179px;">VWR</td>
			<td>89215-218</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">Swingsette Tissue embedding/processing cassette with lid</td>
			<td style="width:179px;">Simport</td>
			<td>M515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Superfrost Plus Slides [size: 25x75x1mm]</td>
			<td style="width:179px;">VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Manual Slide Staining Set</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Staining Dish Green</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Staining Dish White</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4457</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">24-Slide Slide Holder with Detachable Handle</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Oven</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td>6243</td>
			<td>for baking slides at 65 degree</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Dissection microscope</td>
			<td style="width:179px;">Zeiss</td>
			<td>Stemi 2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td style="width:179px;">Nikon</td>
			<td>Eclipse 90i</td>
			<td>Bright and fluoerescent light, with objectives: 10x, 20x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">PAP Pen Super-Liquid Blocker Mini</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>DAI-PAP-S-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Ethanol 200 proof</td>
			<td style="width:179px;">AAPR</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Methanol</td>
			<td style="width:179px;">VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Glacial acetic acid</td>
			<td style="width:179px;">AAPR</td>
			<td>281000ACS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Xylene</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Hydrogen peroxide 25% solution in water</td>
			<td style="width:179px;">ACROS</td>
			<td>202465000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">10% bufered formalin</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>22-026-213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Bovine serum fraction V, heat shock</td>
			<td style="width:179px;">Roche</td>
			<td>3116956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Tween 20</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Sodium citrate</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gavage needle</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">20068-624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rabbit anti Klf5 antibody</td>
			<td style="width:179px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-22797</td>
			<td>Dilution 1: 150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Chicken anti EGFP antibody</td>
			<td style="width:179px;">Millipore</td>
			<td style="width:119px;">AB16901</td>
			<td>Dilution 1: 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rabbit anti Ki67 antibody</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">CRM325B</td>
			<td>Dilution 1: 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Mach3 rabbit AP polymer detection kit</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">M3R533L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Warp red chromogen kit</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">WR806 H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lgr5-EGFP/CreERT2 mice&#160;</td>
			<td>Jackson labs</td>
			<td>008875&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Automated processor</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;">Leica TP1020</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

免疫組織化学染色と組み合わせたスイスの圧延技術は​​、小規模または大規模な腸組織の総合的な分析を可能にします。 C57BL / 6マウス（図1）の大腸のH＆E染色の例は、実現可能性と、この技術の有効性を示す図です。近位、中間、および遠位： 図1に示すように 、画像は、結腸の全ての部分をキャプチャすることが可能です。...

Watch this Scientific Journal Video about Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses at JoVE.com

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

腸の粘膜内層に沿って上皮細胞の正確な同定と位置が異なる細胞系譜を定義することが不可欠です。腸組織の適切な画像化は、最大解像度でのタンパク質発現パターンの同定のために重要です。本研究では、マウスの腸組織を処理するための最適な方法および条件を線引きすることを目指しています。

免疫組織化学および免疫蛍光分析のための腸組織の調製のための改良されたスイス・ローリングテクニック

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Research

JoVE Journal

Biology

Pressure-polishing Pipettes for Improved Patch-clamp Recording

改善されたパッチクランプ記録のための圧力 - 研磨ピペット

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

一次組織の二つのチミジン類似体の免疫蛍光検出（CldUとIDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

プロテオーム解析のためのマウスの硝子体と網膜の内臓摘出

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

An Immunofluorescent Method for Characterization of BarrettÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Esophagus Cells

バレット食道の細胞の特徴のための免疫蛍光法

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

レンチウイルス形質導入と腸オルガノイドの下流分析のためのプロトコル

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

ハイブリッドカット：手に負えない植物組織試料のための改善されたセクショニング方法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

気孔テープ剥離: 細胞試料の調製法の改良

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

-オミクス分析と原発細胞培養のためのコウモリの組織採取

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

24ウェル組織培養プレート(EAgaLプレート)を用いた Aedes aegypti のフェクアンディティと不妊治療アッセイの改善

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

半翅目腸内における植物ウイルスおよび昆虫ベクタータンパク質の免疫蛍光標識

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...