この作品はのGeneralitat de Catalunyaの（2005 - SGR00437）によってサポートされていました。著者は、彼の技術支援のためにJ.ブランコとF.ヴィダルその監督と励ましのため、およびJ.ルーカスに感謝。

I.サンプルの処理および細胞の固定<ol><li>液状化するまで、室温で20分間滅菌した容器に精液のサンプルのままにしておきます。 </li><li>遠心チューブに試料を移し、5分間千グラムで、それを回転させる。 </li><li>静かにパスツールピペットを用いて上清を除去して捨てる。 </li><li> 10mlの最終容量を得るためにボルテックスで混合しながら、低張液（塩化カリウム、0.075M）37予備加熱° C、滴ずつ、追加してください。 </li><li> 37水浴中にチューブを置きます℃で30分間。 </li><li> 5分間千グラム間遠心する。慎重にペレットを乱すことなく、デカンテーションで上澄み液を捨てる。 </li><li> 8ミリリットルの最終的な音量を得るためにボルテックスで混合しながら滴ずつ：ペレットを再懸濁させた後、作りたてのカルノア固定液（酢酸3時01メタノールの）追加。 </li><li>ホワイトペレットを得るために必要な回数だけポイント6と7を繰り返します。 </li><li>作りたてのメタノール追加：酢酸（3:1）（小さなペレットとそのような場合で広がり良好な細胞を得るために必要な細胞濃度に最終的な音量を調整するために滴ずつを、数滴は高い金額でのサンプルに対し、十分になります回収した精子から、最後のボリュームは）1-2 mlを達することができる。テストスライドは、油を塗っていないスライドに再懸濁したサンプルを滴下し、位相差顕微鏡下でそれを調べることによって行うことができます。これは、得られた細胞の分散をチェックし、必要に応じて、さらにスライドで、、それを訂正できるようになるようになる。 </li><li>拡張子を、40cmの高さの最小値からドロップunfrostedのスライドの中央に細胞懸濁液の二、三滴（以前に、それらを脱脂するために-20℃でメタノールに保存されている）。を作るためにプロトコル全体にサンプルの場所を有効にするには、ダイヤモンドの鉛筆を使ってスライドの反対側の精子の拡張子を含む領域をマーク。 </li><li>少なくとも24時間、-20 ° Cでスライドを保存する。 注意：メタノールの添加：酢酸（3:1）滴ずつ混合が精子凝集体の形成を避けるために非常に重要なステップですが。 </li></ol> II。脱凝縮<ol><li>スライドは、-20℃で保存は、（室温でそれらを残す）プロトコルを続行するために解凍する必要があります。 </li><li> 3分ごとに2倍の生理食塩水、クエン酸ナトリウム溶液（2xSSC）の2つの連続した​​コプリンジャーにスライドを配置。 </li><li>各コプリンジャーで2分間エタノール洗浄の一連のスライドを転送する。 70％エタノールで開始、100％で90％、仕上げによって従ってください。室温でそれを残してスライドを乾燥させます。 </li><li> 37℃でジチオスレイトール溶液中でスライド（1,4 - ジチオスレイトール5mmの1％トリトンX - 100、2 - アミノ-2 - （ヒドロキシメチル）-1,3 - プロパンジオール50mmのを）インキュベートしますインキュベーターでCは、クロマチンをdecondenseする。この製品は、精子核のプロタミンのジスルフィド架橋そのコイルDNAを破壊することによって動作します。精子核のDNAは高度に圧縮されるので、これは非常に重要なステップです。ジチオスレイトール溶液（DTT）のスライドのインキュベーション時間は、この製品に対するサンプルの反応性に応じて調整する必要があります。それは2〜15分ごとに異なる​​ことができますが、通常、この時間は、8分程度です。 </li><li>すぐに、各コプリンで3分間2xSSC二つの連続した​​コプリンジャーにスライドを転送する。 </li><li>各コプリンジャー（70％、90％および100％エタノール）で2分間エタノール洗浄の一連のスライドを配置することによって継続する。スライドを室温で乾燥させて。 注：あまりにも短い露光には、いくつかのシグナルの欠如につながるだろう、一方DTTに過度の暴露は、手順の最後にFISHシグナルを分散させるという結果に。 </li></ol> III。交配<ol><li> 73行でホルムアミド溶液（70％Formamide/2xSSC）℃で5分間でコプリンジャーにスライドをインキュベートすることにより、精子のDNAを変性させる。 </li><li>コプリンジャー当たり1分（70％エタノールで85％を開始し、100％で終了）のためにエタノール溶液を介してスライドを転送する。室温で乾燥させるスライドにしておきます。 </li><li>の直接5リットルを追加し、対応するすぐに使用できる15x15カバースリップ（AneuVysionアッセイマルチカラープローブパネル：CEP 18/X/YまたはLSI 21分の13）にプローブの混合物を。 </li><li>それはビデオに示されているように、慎重に標的領域とプローブの混合物をまとめるためにカバースリップの上にスライドを配置。ラバーセメントでそれを封印。 </li><li>予め温めておいた37にスライドを配置° Cハイブリダイゼーションチャンバー（HYBrite™）および6〜24時間37℃で反応させる。 注：精子のサンプルの変性が必須であるのに対し、プローブを変性の必要性は、製造業者によって決定されます。このプロトコルで使用されるプローブの混合物は、使用する準備ができていると、このケースでは、プローブの変性は不要です。 プローブの混合物の体積は、ハイブリダイズした領域のサイズに応じて異なります。 </li></ol> IV。洗浄ポストハイブリダイゼーション<ol><li>ハイブリダイゼーションチャンバーからスライドを取り出してください。 </li><li>ラバーセメントを取り外し、慎重に側に静かにスライドカバースリップを引き出します。 </li><li>排除する非特異的ハイブリダイゼーションシグナルは、NP - 40は73で予め温めておいた0.4xSSC/0.3％でコプリンジャーに2分間、スライドを配置℃の水浴でC。 </li><li> 2xSSC/0.1％のNP - 40 1分間室温で洗浄溶液にスライドを移し。スライドを室温で乾燥させて。 </li><li>ターゲット領域（18 × 18カバースリップ用8リットル）に対比製品を追加。使用される最も一般的な製品は、4&#39;、6 - ジアミジノ-2 - フェニルインドール（DAPI、バイシス社）です。 </li><li>それはビデオに示されているように、カバーガラス（カバーガラスをマニキュアで密封することができるハイブリダイゼーションを維持するために）とハイブリダイズした領域をカバーする。 </li><li>彼らの見通しの分析まで暗所で-20℃でハイブリダイズしたスライドを保存する。これらの条件下でのスライドは、蛍光シグナルの強度を大幅に失われることなく、12ヶ月間まで保存できます。 注：反対は、非特異的プローブのハイブリダイゼーションを洗い流すだろうに対し、より高い温度または過度の洗浄時間は、いくつかの信号の除去をもたらすことができる。 追加された対比製品の体積がカバーするハイブリダイゼーション領域のサイズに応じて異なります。 </li></ol> V.の可視化準備はDAPI /テキサスレッド/ FITCとシングルバンドアクア、FITCおよびテキサスレッドのためのフィルタのためのトリプルバンドフィルターを備えた蛍光顕微鏡下で評価することができます。標準の評価基準は、精子の核1の正しい評価のために従う必要があります。 染色体X、Y、18用のプローブとハイブリダイズする準備は、これら3つの染色体のための信号が表示されます。すべての正常な精子一青の信号（18番染色体に相当する）、そして緑の信号（X -ベアリングの精子）や赤の信号（Y -ベアリング精子を）示す必要があります。 染色体13および21、13番染色体と21番染色体は赤色信号のための緑の信号のためのプローブとハイブリダイズする準備のためにすべての通常の精子に区別されなければなりません。

<ol>
	<li>Blanco, J., Egozcue, J., Vidal, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+chromosome+21+disomy+in+human+spermatozoa+as+determined+by+fluorescent+in-situ+hybridization.">Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in-situ hybridization.</a> Hum. Reprod. 11, 722-726 (1996).</li></ol>

The authors have nothing to disclose.

このプロトコルは、精子のFISHの準備を取得するために人間の精液のサンプルを処理する方法について説明します。それがこのプロトコルを使用すると、雄の配偶子の染色体異常を分析することが可能です。精子異数性のスクリーニングは、基礎研究の文脈でと不妊男性に与えられた生殖アドバイスのいずれかのアプリケーションを持っています。臨床診断に最も広く研究されている染色体は...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1,4-Dithiothreitol (DTT)</td>
<td>Material</td>
<td>Roche</td>
<td>10197777001</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
<td>Material</td>
<td>Roche</td>
<td>10708976001</td>
<td>&nbsp;</td>
<tr>
<td>Acetic acid</td>
<td>Material</td>
<td>Merck</td>
<td>100.063</td>
<td>&nbsp;</td>
<tr>
<td>AneuVysion&reg; Multicolor DNA Probe Kit<ul style="padding:2px;"><li>20xSaline-Sodium Citrate</li><li>4,6-diamidino-2-phenylindole (DAPI II)</li><li>CEP 18/X/Y DNA probe</li><li>NP-40</li><li>LSI 13/21 DNA probe</li></ul></td>
<td>Material</td>
<td>Vysis Inc.</td>
<td>32-161075 <ul style="padding:2px;"><li>30-805850</li><li>30-804941 </li><li>30-171077</li><li>30-804820</li><li>30-171078</li></ul></td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge tube</td>
<td>Material</td>
<td>Nunc</td>
<td>347856</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (glass)</td>
<td>Material</td>
<td>Barloworld Scientific</td>
<td>Hellendahl (ZCT278)</td>
<td>&nbsp;</td>
<tr>
<td>Coplin jar (plastic)</td>
<td>Material</td>
<td>Deltalab</td>
<td>191087</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (15x15)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600115</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips (18x18)</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4600118</td>
<td>&nbsp;</td>
<tr>
<td>Diamond pencil</td>
<td>Material</td>
<td>Hammacher</td>
<td>335117010</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>Material</td>
<td>Merck</td>
<td>100.983</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>Material</td>
<td>Roche</td>
<td>11814320001</td>
<td>&nbsp;</td>
<tr>
<td>Freezer</td>
<td>Tool</td>
<td>Zanussi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gloves</td>
<td>Material</td>
<td>Sanyc</td>
<td>101/3300</td>
<td>&nbsp;</td>
<tr>
<td>HYBrite&trade;</td>
<td>Tool</td>
<td>Vysis Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Immersion Oil</td>
<td>Material</td>
<td>Olympus</td>
<td>35505</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>Tool</td>
<td>Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>Material</td>
<td>Merck</td>
<td>106.009</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette</td>
<td>Material</td>
<td>Labsystems</td>
<td>Finnpipette (4500000)</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette replacement tip</td>
<td>Material</td>
<td>Daslab</td>
<td>16-2001</td>
<td>&nbsp;</td>
<tr>
<td>Nail varnish</td>
<td>Material</td>
<td>Quo-cosmetics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus BX60 epifluorescence microscope</td>
<td>Tool</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.</td>
</tr>
<tr>
<td>Pasteur Pipette</td>
<td>Material</td>
<td>Rubilabor</td>
<td>211.0230</td>
<td>&nbsp;</td>
<tr>
<td>Phase contrast microscope</td>
<td>Tool</td>
<td>Nikon Diaphot</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic pipette (3ml)</td>
<td>Material</td>
<td>Deltalab</td>
<td>200007</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride (KCl)</td>
<td>Material</td>
<td>Fluka</td>
<td>60128</td>
<td>&nbsp;</td>
<tr>
<td>Rubber cement</td>
<td>Material</td>
<td>Best-test</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>Material</td>
<td>Knittel gl&auml;ser</td>
<td>4520022</td>
<td>&nbsp;</td>
<tr>
<td>Slide Saver Boxes</td>
<td>Material</td>
<td>Deltalab</td>
<td>19276.1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile container</td>
<td>Material</td>
<td>Deltalab</td>
<td>409726</td>
<td>&nbsp;</td>
<tr>
<td>Syringe</td>
<td>Material</td>
<td>PentaFerte</td>
<td>002022300</td>
<td>&nbsp;</td>
<tr>
<td>Thermometer</td>
<td>Material</td>
<td>Comark Instruments, Inc</td>
<td>KM12</td>
<td>&nbsp;</td>
<tr>
<td>Tri-distilled water</td>
<td>Material</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Material</td>
<td>Sigma</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Material</td>
<td>B/Braun</td>
<td>Aesculap (BD224R)</td>
<td>&nbsp;</td>
<tr>
<td>Vertical laminar flow hood</td>
<td>Tool</td>
<td>Burdinola</td>
<td>OR-ST 1500</td>
<td>&nbsp;</td>
<tr>
<td>Vortex mixer</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>FB15012</td>
<td>&nbsp;</td>
<tr>
<td>Water bath</td>
<td>Tool</td>
<td>Raypa&reg;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence in situ hybridization (fish) protocol in human sperm

Aneuploidiesは、ヒトで最も頻繁に染色体異常です。これらの異常のほとんどは、両親のgametogenic過程で減数分裂のエラーに起因する。人間の男性では、これらのエラーは、子孫にこれらの異常を送信するリスクの増加を表す数値染色体異常を持つ精子の生産につながることができます。 このような理由から、精子の核のin situハイブリダイゼーション（FISH） の蛍光の技法は、広く臨床診断のコンテキストに組み込まプロトコルになっています。この練習では、遺伝的生殖アドバイスを求める患者の配偶子の数値染色体異常の頻度の推定値を提供します。 日付に、最も頻繁に精子のFISH分析に含まれる染色体は染色体X、Y、13、18および21です。 このビデオの記事は、順を追って説明し、人間の精液のサンプルを処理し、解決する方法、精子のクロマチンをdecondenseと変性する方法、どのように精子FISHの準備を取得するために進むために、そして顕微鏡での結果を可視化する方法。最も関連性の高い手順の特記事項は、最高の結果を達成するために与えられている。

Watch this Scientific Journal Video about Fluorescence in situ hybridization FISH Protocol in Human Sperm at JoVE.com

Fluorescence in situ hybridization FISH Protocol in Human Sperm

このビデオの記事では、良質の蛍光を達成するために精液のサンプルを処理する方法を順を追って、説明します<em&gt;その場で</em人間の精子で&gt;ハイブリダイゼーション。得られた製剤は、臨床診断のコンテキストで異数スクリーニングに用いることができる。

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 ...

fluorescence-in-situ-hybridization-fish-protocol-in-human-sperm

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

30.0K ビュー.  Universitat Autónoma de Barcelona. このビデオの記事では、良質の蛍光を達成するために精液のサンプルを処理する方法を順を追って、説明しますその場でハイブリダイゼーション。得られた製剤は、臨床診断のコンテキストで異数スクリーニングに用いることができる。

ビデオ: Fluorescence in situ hybridization FISH Protocol in Human Sperm

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性その場で</emノルウェースプルースと植物種の範囲に適用>ハイブリダイゼーションプロトコール

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

生物学

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 ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNAの蛍光による単一細胞遺伝子転写の解析<em&gt; in situで</em&gt;ハイブリダイゼーション（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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 ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

組み合わせたDNA-RNA蛍光<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）差別雌マウス胚性幹細胞におけるX染色体の不活性化を研究するために、

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

クイック蛍光<em&gt;その場で</emX染色体不活性化におけるヒストン修飾の免疫蛍光とのXist RNA複合のための&gt;ハイブリダイゼーションプロトコル

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

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.

蛍光DNAための簡単​​な方法<em&gt;その場で</em&gt;ぺしゃんこ染色体へのハイブリダイゼーション

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

蛍光を用いて観察し、テロメアの定量化と反復配列<em&gt;その場で</emPNAプローブのある&gt;ハイブリダイゼーション（FISH）<em&gt;線虫（Caenorhabditis elegans）</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

蛍光その場でハイブリダイゼーション（FISH）プロトコル