MTの研究室では、国立科学財団（DBI - 0820610およびIOS - 1022102）と健康のニューヨーク州省（NYSTEM - C024308）からの補助金によってサポートされています。 CMは、教育と科学（200​​7〜0937）と財団ラファエルデルピノ、スペインのスペイン省からポスドクを受けた。

<ol>
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利害の衝突は宣言されません。

ここで概説したin situハイブリダイゼーション法偉大な携帯電話の解像度、高感度および特異性に関心の任意の遺伝子の時空間発現パターンの直接可視化することができます。プロトコルは、遺伝子間の発現レベルの定量的な比較を許可しません。しかし、法の高感度と分解能は、遺伝子発現を解析の大部分の他の方法では不可能な組織8、18、内遺伝子発現の勾配を明らかにすることができます。 プロトコルは、トラブルシューティングが問題にすることができます多くの手順が含まれています。プロトコルの最も重要なステップは、組織の固定とプローブの選択とラベリングです。低DIGの取り込みと不完全浸透し組織とプローブは、バックグラウンドの上に検出することが困難になる可能性のある非常に微弱な信号が得られます。興味の各々の新しい遺伝子の場合、それはdiffeから派生したいくつかの異なるプローブを試してみることをお勧め転写産物の領域を借りる。時折、遺伝子の別の小さな領域を標的とする二、三のプローブが強いと、特定のハイブリダイゼーションシグナルを得るために組み合わせることが必要な場合があります。さらに、そのような温度およびハイブリダイゼーションバッファーの組成などのハイブリダイゼーション条件は、、それぞれの遺伝子またはハイブリダイゼーションのストリンジェンシーを下げるか上げるために組織を最適化することができます。また、プロテアーゼ処理のステップは、変数であり、異なる植物種のためにまたは非常に低濃度と転写産物を検出するためのプロトコルを適合させるために変更することができます。両方陽性および陰性コントロールプローブの包含は、プロトコルの問題のポイントを識別する助けになるでしょう。 手順は、パラフィン包埋植物の組織切片用が若干変更して最適化されてin situハイブリダイゼーションホールマウントにも使用できます。広く動物研究19で使用されているが、in situハイブリダイゼーション全体のマウントはLになりますこのような胚、根や若い分裂組織20,21として簡単に潜入することができるの植物組織、にimited。プロトコルは、簡単に同時に二つの異なるmRNAを検出したり、蛋白質15,22,23と一緒に成績証明書をローカライズするために変更することができます。最後に、それは、植物におけるsmall RNAの発現パターンの可視化には、このメソッドのアプリケーションを拡張することが可能です。 miRNAの発現パターンは、トウモロコシとシロイヌナズナ8,14の両方でこのプロトコルを使用して決定されている。さらに最近では、in vitro転写されたプローブで 、市販のロックされた核酸（LNA）信号の感度18、24を増加させるオリゴプローブのDIG標識に置き換えられています。

<table border="1"><tbody><tr><th>製品</th><th>会社</th><th>カタログ番号</th><th>解説</th></tr><tr><td> Cytoseal 60 4オンス</td><td>フィッシャー</td><td> 23-244-256 </td><td>トルエンベースの8310から4 </td></tr><tr><td> Histoclear </td><td>フィッシャー</td><td> 50-899-90147 </td><td></td></tr><tr><td>組織パスParaplastプラス</td><td>フィッシャー</td><td> 23-021-400 </td><td></td></tr><tr><td> N +ナイロンメンブレンハイボンド- N + </td><td> GEヘルスケア</td><td> RPN203B </td><td></td></tr><tr><td> RNaseのOUT、40 U /μL </td><td>インビトロジェン</td><td> 10777019 </td><td></td></tr><tr><td> RQ1 RNaseフリーのDNase、1 U /μlの</td><td>プロメガ</td><td> M6101 </td><td></td></tr><tr><td> DIGラベルされたコントロールRNA </td><td>ロッシュ</td><td> 11585746910 </td><td></td></tr><tr><td> DIG RNAラベリングミックス</td><td>ロッシュ</td><td> 11277073910 </td><td></td></tr><tr><td> SP6 RNAポリメラーゼ1,000 U </td><td>ロッシュ</td><td> 10810274001 </td><tD&gt; </td></tr><tr><td> T3 RNAポリメラーゼ1,000 U </td><td>ロッシュ</td><td> 11031163001 </td><td></td></tr><tr><td> T7 RNAポリメラーゼ1,000 U </td><td>ロッシュ</td><td> 10881767001 </td><td></td></tr><tr><td>のRNase </td><td>ロッシュ</td><td> 109142 </td><td>グリセロール50パーセントトリス100mMのpH8.0で溶解する</td></tr><tr><td>ブロッキング試薬</td><td>ロッシュ</td><td> 1 096 176 </td><td> ° C TBSバッファーで70までの耐熱</td></tr><tr><td>羊150 Uから抗ジゴキシゲニン- AP、Fabフラグメント</td><td>ロッシュ</td><td> 1 093 274 </td><td></td></tr><tr><td> NBT / BCIPストック溶液</td><td>ロッシュ</td><td> 1 681 451 </td><td></td></tr><tr><td>ミニクイックスピンRNAカラム</td><td>ロッシュ</td><td> 11814427001 </td><td></td></tr><tr><td> E.からtRNAの大腸菌 </td><td>ロッシュ</td><td> 109541 </td><td></td></tr><tr><td>トリトン® X - 100 </td><td>シグマ</td><td> T8787 </td><td></td></tr><tr><td> Tween - 20を</td><td>シグマ</td><td> P9416 </td><td></td></tr><tr><td>脱イオン化ホルムアミド</td><td>シグマ</td><td> F9037 </td><td></td></tr><tr><td> 放線griseusタイプXIVからのプロテアーゼ</td><td>シグマ</td><td> P5147 </td><td> 37 MilliQ水とプレダイジェストで希釈して4時間° C </td></tr><tr><td>トリエタノールアミン</td><td>シグマ</td><td> 90279 </td><td>水で希釈し、HClでpH8.0にもたらす</td></tr><tr><td>無水酢酸</td><td>シグマ</td><td> A6404 </td><td></td></tr><tr><td> ロイコノストック属からデキストラン硫酸のナトリウム塩。 </td><td>シグマ</td><td> D8906 - 5G </td><td>デキストラン硫酸℃で溶解するために80にまで加熱する必要があります。 </td></tr><tr><td>デンハルト溶液の50倍の濃縮</td><td>シグマ</td><td> D2532 </td><td></td></tr><tr><td>ウシ血清からアルブミン - ≥98％ </td><td>シグマ</td><td> A7906 </td><td></td></tr><tr><td>エオシンY二ナトリウム塩</td><td>シグマ</td><td> E4382 </td><td>飽和するまで96〜100％エタノールに溶解する</td></tr><tr><td>パラホルムアルデヒド</td><td>シグマ</td><td> P6148 </td><td></td></tr><tr><td>エタノール絶対200プルーフ</td><td></td><td></td><td></td></tr><tr><td>フェノール/クロロホルム</td><td></td><td></td><td></td></tr><tr><td>ベース型</td><td>電子Microsocpy科学</td><td> 62352〜15 </td><td></td></tr><tr><td>埋め込みリング</td><td>フィッシャー</td><td> 22-038-197 </td><td></td></tr><tr><td>キャップと20ミリリットル使い捨てシンチレーションバイアル、 </td><td> VWR </td><td> 66020-326 </td><td></td></tr><tr><td>プローブオンプラススラ​​イド</td><td>フィッシャー</td><td> 22-230-900 </td><td></td></tr><tr><td>マイクロカバーガラス24x50ミリメートル</td><td> VWR </td><td> 48404-452 </td><td></td></tr><tr><td>ワットマン濾紙3ミリメートル</td><td> VWR </td><td> 89022-360 </td><td></td></tr><tr><td>三ガラス皿一STAInlessスチールスライドラック</td><td>電子Microsocpy科学</td><td> 71420〜25 </td><td>二つのガラスはhistoclear治療のために必要なくぼみと最後のものは、無水酢酸の治療のために使用されています</td></tr><tr><td> 18清潔なプラス​​チック容器</td><td>電子Microsocpy科学</td><td> 71402 </td><td></td></tr><tr><td>密閉式蓋つきの2つまたは3つの大型フラットプラスチックの箱</td><td></td><td></td><td>ハイブリダイゼーションおよび抗体の手順では必要</td></tr><tr><td>ファインブラシ</td><td></td><td></td><td>ワックスリボンを渡すのに役立ちます</td></tr><tr><td>ミクロトーム</td><td></td><td></td><td>ライカ</td></tr><tr><td>セルフクリーニングドライ真空システム</td><td>ウェルチ</td><td> 2025 </td><td></td></tr><tr><td>ウォーマーをスライドさせ</td><td>フィッシャー</td><td></td><td></td></tr><tr><td>加熱ブロック</td><td></td><td></td><td> 60 ° Cおよび85 ° Cで必要</td></tr><tr><td> 58〜60時インキュベーター° C </td><td>フィッシャー</td><td></td><td>溶融Paraplastと手順について</td></tr><tr><td> 55 ° Cと37 ° Cのオーブンまたは水浴</td><td>フィッシャー</td><td></td><td>温度の間にクイックチェンジによる2を必要とする</td></tr><tr><td>遠心（4 ° C - 20 ° C） </td><td></td><td></td><td></td></tr><tr><td>ドラフト</td><td></td><td></td><td></td></tr></tbody></table>

in situ hybridization for the precise localization of transcripts in plants

過去十年間のゲノム研究の進歩に伴い、植物の生物学は、遺伝子発現の大規模な定量的な分析を提示する多くの研究を見ている。マイクロアレイや次世代シーケンシングのアプローチは、発達、生理、ストレス応答のプロセスを調査エピジェネティクスとsmall RNAの経路を分析、及び1-3大規模な遺伝子調節ネットワークを構築するために使用されている。これらの技術は大規模な遺伝子セットの同時分析を容易にする一方で、彼らは一般的に遺伝子発現の変化の非常に限られた時空間解像度を提供しています。この制限は、部分的に4-7をソートlasermicrodissectionまたは蛍光活性化細胞と一緒にどちらかのプロファイリングメソッドを使用して克服することができます。しかし、完全に遺伝子の生物学的役割を理解するために、細胞の解像度での表現のその時空間パターンの知識が不可欠です。特に、開発や環境へのSTIの影響を研究するときmuliと変異体は、遺伝子の発現パターンの詳細な分析が不可欠になることができます。特定の細胞型における発現の損失または利得に関連付けられているときに例えば、キー調節遺伝子の発現レベルの微妙な量的な違いは、劇的な表現型につながることができます。 いくつかの方法は、日常的に遺伝子発現パターンの詳細な検査のために使用されています。一つは、トランスジェニックレポーターラインの分析を通じてです。このような分析は、しかし、複数の遺伝子を解析または変換に負えない所で作業する際に時間がかかるになることができます。また、導入遺伝子の発現パターンは内因性遺伝子のことを模倣することを保証するための独立した検証は、通常は必要です。 in situハイブリダイゼーション免疫組織化学的蛋白質の局在またはmRNAは、細胞や組織内の遺伝子発現の直接可視化するための比較的高速な代替案を提示。後者は、それが容易にuをすることができる明確な利点を持っています興味のある任意の遺伝子上のsed。situハイブリダイゼーションでは、目的の遺伝子のin vitro転写により得られたプローブを標識したアンチセンスRNAとのハイブリッド形成による細胞内で標的mRNAの検出を可能にする。 ここでは、非常に感度と特異的であり、植物における遺伝子発現の in situ局在のためのプロトコルの概要を示します。それは、パラホルムアルデヒド固定、パラフィン包埋切片、組織学の優れた保全を与え、そして免疫検出し、アルカリフォスファターゼ比色反応により可視化DIGラベルされたプローブでの使用に最適化されています。このプロトコルは、正常な植物種の広い範囲から採取した組織の数に適用されている、とmRNAと同様の低分子RNA 8月14日の表現を分析するために使用することができます。

Watch this Scientific Journal Video about In Situ Hybridization for the Precise Localization of Transcripts in Plants at JoVE.com

In Situ Hybridization for the Precise Localization of Transcripts in Plants

市販<em&gt;その場で</emここで説明する&gt;ハイブリダイゼーションプロトコールは、高感度と特異性を持つ細胞レベルでのmRNAと小さなRNAの発現の直接局在することができます。手順はパラフィン包埋植物の組織切片用に最適化され、植物や組織の広い範囲に適用可能である、と10日以内に完了することができます。

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 ...

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JoVE Journal

Biology

In Situ Hybridization for the Precise Localization of Transcripts in Plants

51.2K ビュー.  Cold Spring Harbor Laboratory. 市販その場でハイブリダイゼーションプロトコールは、高感度と特異性を持つ細胞レベルでのmRNAと小さなRNAの発現の直接局在することができます。手順はパラフィン包埋植物の組織切片用に最適化され、植物や組織の広い範囲に適用可能である、と10日以内に完了することができます。

ビデオ: In Situ Hybridization for the Precise Localization of Transcripts in Plants

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 ...

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

ホールマウントRNAを蛍光<em&gt;その場で</emの&gt;ハイブリダイゼーション<em&gt;ショウジョウバエ</em&gt;胚

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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 ...

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 ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

全体のマウント<em&gt;その場で</em腹足類軟体動物のための&gt;ハイブリダイゼーション法<em&gt;モノアラガイstagnalis</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.