この作品は、米国国立衛生研究所HL082798とHL111580を補助金によって支えられている。

1。腎臓組織切片<ol><li>ホルマリン固定した（10％）パラフィン包埋腎臓切片をスライドは、分析の前に数ヶ月間保存することができるMicrom社HM 355 S.を用いて厚さ5mmの顕微鏡スライド上に切断される。 </li></ol> 2。溶液の調製<ol><li>オートクレーブのスクリュートップボトルに蒸留H 2 Oに1X PBSを1 Lを用意します。のddH 2 Oを1Lで別のボトルを埋める各ボトル、カバーにDEPCの1ミリリットルを追加し、激しく振る。ソリューションは、RNA分解酵素を破壊した後、オートクレーブを室温で一晩放置します。 PBS-Tを作るために1X PBS 1Lに400ミリリットルのTween-20を追加します。 </li><li>プロテイナーゼKバッファー（50のトリス-塩酸、pHが8.0と、DEPCでの1のCaCl 2処理水）を準備します。 </li><li> PBS-Tで0.2％グリシン溶液を調製する。 </li></ol> 3。組織標本<ol><li> 50％ホルムアミド、5×SSC、0.1％のTween、9.2からなるハイブリダイゼーション緩衝液（50 ml）を大量調製pHが6、50μg/ mlのヘパリン、およびDEPC 500μg/ mlの酵母RNAへの調整のためのクエン酸は、蒸留H 2 Oを扱うC.度-80で1ミリリットルずつ、ストアに分ける</li><li> 5分ごとに新鮮なキシレンに3回洗浄することによって、組織からパラフィンを除去。 </li><li>のddH 2 Oに（100％、75％、50％、および25％）エタノールの濃度を減少させることで、5分間のインキュベーションによって、組織切片を再水和</li><li>完全に1分間、組織切片（約0.4〜0.5ミリリットル）をカバーするのに十分なDEPCでスライドを扱う。組織が乾燥しないようにしてください。 </li><li> DEPCのスライドが適切な処分のための廃棄物を含むDEPCを集め、5分間二回、PBS-Tをあしらったすすぎます。すべての試薬は、DEPCこの時点からRNA分解酵素を除去するために扱われるべきである。 </li><li>予熱し、プロテイナーゼK緩衝液および10μg/ mlの最終濃度にプロテイナーゼKを加える。プロテイナーゼK溶液で組織切片をカバーし、5分間37℃でインキュベートする。 </li><li>迅速proteinasを削除電子K溶液で30秒間PBS-T中の0.2％のグリシンを加える。 </li><li> DEPCでのすすぎのスライドを30秒ごとに二回、PBS-Tを治療した。 </li><li> 10分間焼きたての4％パラホルムアルデヒド中でのセクションを修正します。 </li></ol> 4。混成<ol><li>組織切片あたりの50のハイブリダイゼーション緩衝液（50％ホルムアミド、5×SSC、0.1％のTween、9.2 mMのpHを6に調整するためのクエン酸、50μg/ mlのヘパリン、500μg/ mlの酵母RNA）を追加し、RNA分解酵素フリーのHybriSlipsでカバー組織切片よりちょうど大きくカット。 </li><li> 3 &#39;ジゴキシゲニン標識プローブ（プローブの通常のDNAのTmのために決定ハイブリダイゼーション温度で2時間、湿度制御、ハイブリダイゼーションオーブンでプレハイブリダイゼーション切片-21°C〜、のmiR-382のために（ すなわち 54℃で、DNAのTm = 75℃を探る）、チャンバーを維持するために50％formamide/50％5×SSCに浸したティッシュラボワイプを使用して加湿。 </li><li> miRNAプローブ、ポジティブコントロール（U6、核内低分子RNA）とネガティブコントロール（スクランブルを希釈ハイブリダイゼーション緩衝液中の40 nmの配列）（バッファの75μl）をセクションごとに必要とされる。 </li><li> 5分間65度Cで、ハイブリダイゼーション緩衝液中のプローブを加熱する。 </li><li>ハイブリダイゼーションオーブンからスライドを削除し、プレハイブリダイゼーションからカバースリップと過剰ハイブリダイゼーション緩衝液を除去します。 </li><li>直接組織に、組織切片あたりのプローブを含有する緩衝液の75μlを加える。組織切片をカバーするのに十分な大きHybriSlipsで組織を覆う。 </li><li>スライドホルダーレベルを維持し、ステップ4.2（約16時間）の温度で一晩のインキュベーションのためのハイブリダイゼーションオーブンに戻ります。 </li></ol> 5。ジェンシーウォッシュ<ol><li>組織からカバースリップを解放するに役立つだけでカバースリップの1辺の下で、ハイブリダイゼーション温度に加熱し、200μlの2×SSCを追加します。スライドを傾けてカバースリップを取り除きます。 </li><li> 30分ごとにハイブリダイゼーション温度の3倍で、50％ホルムアミド、50％、2×SSC200μlのスライドを洗浄します。 </li><li>すすぎスライドは低速でオービタルシェーカー上で5分間ずつ室温でPBS-Tで5倍。 </li></ol> 6。検出<ol><li>低速でオービタルシェーカー上で室温で1時間、緩衝液（2％ヤギまたはウマ血清、2 mg / mlのPBS-TでBSA）でブロッキングスライドをインキュベートする。 </li><li> 1:100でブロッキング緩衝液中で、抗DIG-AP Fab断片を希釈する。組織切片当たり100μlを追加し、セクションをカバーするのに十分な大きカットHybriSlipsでカバーしています。 </li><li> 4℃で一晩（約16時間）の加湿チャンバー内で抗体をインキュベートする。 </li><li>低速でオービタルシェーカー上で5分間ずつ、PBS-T 7Xでスライドを洗浄します。 </li><li> 5分間ずつ、AP緩衝液（100mMトリスHCl pHは9.5、50のMgCl 2、100mMのNaClを、0.1％Tween 20）で3回スライドを洗浄します。 </li><li> APバッファにNBT / BCIP溶液を追加し（200 mlの緩衝液μl/10） </li><li>完全にすべての組織切片をカバーするために、各スライドに希釈し、NBT / BCIP溶液を400〜500ミリリットルを追加します。暗い、レベル、humidifで発展4-5時間のためにIED室。 </li></ol> 7。マウントスライド<ol><li>各5分間のエタノール濃度（25％、50％、75％、100％）を増加させるのセクションを脱水する。 </li><li>のセクションをクリアするためにキシレン5Xでインキュベートする。 </li><li>マウントはパーマウント封入剤と乾燥一夜にスライドする。 </li></ol>

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開示することは何もありません。

この記事の目的は、ホルマリン固定腎臓の組織でうまく機能situハイブリダイゼーションにおける miRNAのためのプロトコルを記述することでした。このプロトコルを作業中に染色アーティファクトのいくつかの重要な源が特定されている。これらの点に十分注意し、染色アーティファクトを回避し、成功したISHの実行の可能性を高めることができます。 組織?...

マイクロRNAは、内因的に産生されている非コードRNA（約22ヌクレオチド長）小さい。これらは、一般に翻訳抑制又はmRNA分解を介してタンパク質発現を抑制するように機能する。 miRNAは、それが可能な1つのmiRNAが複数のターゲットを抑制できるようにすること、不完全な相補性を有するmRNA標的に結合する。 細胞型および構造はmiRNAを表現し理解することは、miRNAの発現の変化が、細胞や組織の表現型に影響を与えるそれを通してのメカニズムを理解することの重要な部分です。そのようなmiRNAの配列決定の方法、定量PCRおよびノー​​ザンブロッティングは、全組織におけるmiRNAの検出のために使用することができるが、このアプローチは、一つは、それらが所定の組織内から来た特定の細胞型を決定することはできません。これらの方法を用いて、分析前に細胞性および構造部品の切開は非常に困難であることができ、十分なアイソレーションを達成するために必要な条件は、文献につながる可能性がRNAの遺伝子発現または劣化terations。 in situハイブリダイゼーションは、組織中のマイクロRNAマイクロRNA位置および発現レベルを可視化するために使用される方法である。この技術は、腎臓などの異質な構造で構成組織では特に貴重である。 マイクロRNAは、腎臓発生の2,3および生理学4において調節的役割を果たしていることが示されている。マイクロRNA発現の変化はまた、線維症5月10日 、糖尿病性腎症7、腎癌11,12、および急性腎障害13などの腎病理に関与することが示されている。我々の研究では、腎臓組織のためのin situハイブリダイゼーションのマイクロRNAの最適化は、健康と病気14の両方におけるmiRNA発現の正確な構造上の位置を決定するための貴重であったことを見出した。管状の異なるマイクロRNAの細胞発現の決定は、TAのためにそれらの調節に重要であるrgetsは細胞機能に依存することができる。疾患状態では、miRNAの発現の変化が機能に影響を与えることができる方法を決定することも重要です。 ここで説明する方法の目標は、 他 Kloostermanによって開発された既存のISHの方法論に基づいて構築することでした。15、他の研究16,17、およびExiqon社1によって提案されたものと、ホルマリン固定腎臓組織のための方法を最適化します。我々が正常に片側尿管閉塞18で腎マイクロRNAのmiR-382発現における明確な地域差を識別するために、このメソッドを使用している。このアプローチは、追加の最適化、ならびに他の組織とともに使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="481">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

この記事では、マイクロRNAプローブをタグ付けされたジゴキシゲニンを用いたin situハイブリダイゼーションによって、腎臓におけるmiRNAの比色検出のための方法を説明します。もともとExiqon社miRNAプローブ1との幅広い使用にKloostermanらによって開発されたこのプロトコルは、腎臓組織におけるmiRNA解析に固有の課題を克服するように変更されました。これらには、構造同定および残留プローブおよび抗体を除去するのは難しいなどの問題があります。比較的薄いを用い、厚さ5mm、強力なプローブ信号が細胞内に保持しながら、腎臓構造の明確な可視化のために許可された組織切片。加えて、プローブ濃度およびインキュベーション条件は、低いバックグラウンドおよび非特異的シグナルを有するマイクロRNA発現の可視化を容易にするように最適化した。ここで、最適化されたプロトコルは、手順の終わりにスライドを装着を通して初期の組織収集および準備を覆って、記載されている。基本componeこのプロトコルの国税庁は、他の組織および細胞培養モデルに適用するために変更することができる。

ハイブリダイゼーション、インキュベーション中に乾燥させたり、NBT / BCIPの開発中に、より濃く染色結局、一般の手順を洗うようになる組織切片の面積は。1 HybriSlipはのエッジの脱落した腎臓切片の一部を示していますそれは部分的に脱水になることを可能にする組織。残りの手順での再水和とカバレッジにもかかわらず、脱水部分の信号が人為的に高いです。 <...

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

この記事では、ホルマリン固定腎臓切片にマイクロRNA発現の比色検出のために最適化されたin situハイブリダイゼーションプロトコルについて説明します。

マイクロRNA<em&gt;その場で</em&gt;ホルマリン固定腎臓組織のためのハイブリダイゼーション

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

Research

JoVE Journal

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

14.9K ビュー.  Medical College of Wisconsin. この記事では、ホルマリン固定腎臓切片にマイクロRNA発現の比色検出のために最適化されたin situハイブリダイゼーションプロトコルについて説明します。 

ビデオ: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

蛍光その場で</emヒト精子の>ハイブリダイゼーション（FISH）プロトコル

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性その場で</em組織における多様な遺伝子発現パターンを検出するための>ハイブリダイゼーション

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

蛍光灯と組み合わせることで染色体の複製タイミング<em&gt;その場</em&gt;ハイブリダイゼーション

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

ホルマリン固定からプロテオミクスサンプル調製およびパラフィン包埋組織

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

マイクロRNA

要約

さらに動画を探す

この動画の章

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

蛍光その場でハイブリダイゼーション（FISH）プロトコル

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

放射性その場でハイブリダイゼーション

蛍光によるウイルスRNAの検出

蛍光灯と組み合わせることで染色体の複製タイミング

蛍光を用いたmRNA分子の可視化と分析

ホルマリン固定からプロテオミクスサンプル調製およびパラフィン包埋組織

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

蛍光DNAための簡単な方法

マイクロRNA

要約

さらに動画を探す

この動画の章

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

蛍光その場でハイブリダイゼーション（FISH）プロトコル

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

放射性その場でハイブリダイゼーション

蛍光によるウイルスRNAの検出

蛍光灯と組み合わせることで染色体の複製タイミング

蛍光を用いたmRNA分子の可視化と分析

ホルマリン固定からプロテオミクスサンプル調製およびパラフィン包埋組織

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

蛍光DNAための簡単​​な方法

蛍光DNAための簡単な方法