我々は、スピーカーの音声になるボランティアのための私達の映画の音楽を提供し、マイケルエリオットの株式にアンダースBovinに非常に感謝しています。サポートがカールTrygger財団、農業科学、スウェーデン研究評議会（VR）のスウェーデンの大学戦略的研究プログラム農業ゲノム機能（AgriFunGen）、および環境のためのスウェーデンの研究評議会、農業科学、及び空間計画（FORMASによって提供されていました）。

<ol>
	<li>Carr, S. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+homeotic+gene+expression+defines+developmental+arrest+stages+in+Brassica+oleracea+L.+vars.+botrytis+and+italica.">Floral homeotic gene expression defines developmental arrest stages in Brassica oleracea L. vars. botrytis and italica.</a> Planta. 201 (2), 179-188 (1997).</li><li>Coen, E. S., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+war+of+the+whorls:+genetic+interactions+controlling+flower+development.">The war of the whorls: genetic interactions controlling flower development.</a> Nature. 353 (6339), 31-37 (1991).</li><li>Jackson, D., Gurr, S. J., McPherson, M. J., Bowles, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+in+plants.">In situ hybridization in plants.</a> Molecular Plant Patology: a practical approach. , (1991).</li><li>Leitch, A. R., Schwarzacher, T., Jackson, D., Leitch, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization: a practical guide. , (1994).</li><li>Smyth, D. R., Bowman, J. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+flower+development+in+Arabidopsis.">Early flower development in Arabidopsis.</a> Plant Cell. 2 (8), 755-767 (1990).</li><li>Azevedo, H., Lino-Neto, T., Tavares, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+high-quality+RNA+isolation+from+needles+of+adult+maritime+pine+trees.">An improved method for high-quality RNA isolation from needles of adult maritime pine trees.</a> Plant Molecular Biology Reporter. , 333-338 (2003).</li><li>Sundstrom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+active+in+developing+pollen+cones+of+Norway+spruce+(Picea+abies)+are+homologous+to+the+B-class+floral+homeotic+genes+in+angiosperms.">MADS-box genes active in developing pollen cones of Norway spruce (Picea abies) are homologous to the B-class floral homeotic genes in angiosperms.</a> Dev Genet. 25 (3), 253-266 (1999).</li><li>Tandre, K., Svenson, M., Svensson, M. E., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+gene+structure+and+activity+in+the+regulation+of+reproductive+organ+development+of+conifers+and+angiosperms.">Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms.</a> Plant J. 15 (5), 615-623 (1998).</li><li>Winter, K. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MADS-box+genes+reveal+that+gnetophytes+are+more+closely+related+to+conifers+than+to+flowering+plants.">MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants.</a> Proc Natl Acad Sci U S A. 96 (13), 7342-7347 (1999).</li><li>Savard, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloroplast+and+nuclear+gene+sequences+indicate+late+Pennsylvanian+time+for+the+last+common+ancestor+of+extant+seed+plants.">Chloroplast and nuclear gene sequences indicate late Pennsylvanian time for the last common ancestor of extant seed plants.</a> Proc Natl Acad Sci U S A. 91 (11), 5163-5167 (1994).</li><li>Jack, T., Brockman, L. L., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeotic+gene+APETALA3+of+Arabidopsis+thaliana+encodes+a+MADS+box+and+is+expressed+in+petals+and+stamens.">The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens.</a> Cell. 68 (4), 683-697 (1992).</li><li>Bowman, J. L., Smyth, D. R., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+directing+flower+development+in+Arabidopsis.">Genes directing flower development in Arabidopsis.</a> Plant Cell. 1 (1), 37-52 (1989).</li><li>Kramer, E. M., Irish, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+genetic+mechanisms+controlling+petal+development.">Evolution of genetic mechanisms controlling petal development.</a> Nature. 399 (6732), 144-148 (1999).</li><li>Sundstrom, J., Engstrom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conifer+reproductive+development+involves+B-type+MADS-box+genes+with+distinct+and+different+activities+in+male+organ+primordia.">Conifer reproductive development involves B-type MADS-box genes with distinct and different activities in male organ primordia.</a> Plant J. 31 (2), 161-169 (2002).</li><li>Carlsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+altered+expression+of+a+large+number+of+nuclear+genes+in+developing+cytoplasmic+male+sterile+Brassica+napus+flowers.">Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers.</a> Plant J. 49 (3), 452-462 (2007).</li><li>Teixeira, R. T., Farbos, I., Glimelius, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+levels+of+meristem+identity+and+homeotic+genes+are+modified+by+nuclear-mitochondrial+interactions+in+alloplasmic+male-sterile+lines+of+Brassica+napus.">Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus.</a> Plant J. 42 (5), 731-742 (2005).</li><li>Ying, S., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+of+in+situ+hybridization.">The technique of in situ hybridization.</a> Methods in Molecular Medicine. 56, 263-283 (2001).</li></ol>

著者らは、開示することは何もない。

Pの二つの側面トドマツプロトコルはAに比較して最適化されたシロイヌナズナ/ B.セイヨウプロトコル、RNase A処理およびプロテイナーゼKの濃度。 RNaseはバックグラウンド信号を除去し、従って信号の特異性を17増加させる。プロテイナーゼK処理、プローブが入ると興味のRNAのプールにハイブリダイズすることができるように組織を透過するために必要です。 DAL13アンチセンスプローブは、30分（図2D、FおよびH）のためにRNaseで処理のセクションに比べてのRNase処理（図3C、E、G）なしで高いシグナルを与えた。以来のRNase治療は、それがプロトコルから削除された信号を増強しなかった。 1、3および5μg/ mlを、プロテイナーゼK処理は、3つの異なる濃度で試験した。 P.の場合トドマツ 、低または無信号が1μg/ mlのプロテイナーゼK（図3C - D）を用いて観察された。 5μg/ mlのプロテ​​イナーゼKを使用する場合は信号強度に明らかな差が見られなかったので、強い信号は、3μg/ mlの（図3E - F）、5μg/ mlの（図3G - H）プロテイナーゼKの両方が得られた3μg/ mlのに比べて、我々は我々のプロトコルの3μg/ mlを使用することにしました。 P.の高いプロテイナーゼKの濃度の必要可能性が高いですトドマツ男性の円錐Aに比べシロイヌナズナとB.セイヨウ花芽は、フェノール類および多糖類の高いレベルでよりコンパクトな組織によるものです。 固定時のとA.いくつかの違いを埋め込 ​​むシロイヌナズナ/ B.セイヨウプロトコルとP.トドマツプロトコルは明白です。興味の組織はRNAの保持を提供するために固定されており、不十分な固定材料はmRNAが非常に豊富であるにもかかわらず、低いか検出できないシグナルを与えるかもしれないので、これは重要なステップです。組織は、クリアと組織の損傷を避けるために、漸進的な変化のワックスに埋め込 ​​まれ、脱水され、そしていくつかのプロトコル0.85パーセントNaClにさらにセル3の収縮や腫れを防ぐために脱水エタノールに追加されます。埋め込み時の脱水工程は、最低でRNase活性を保つために氷の上で行うことができます。 P.でトドマツプロトコル4％パラホルムアルデヒド固定ソリューションは、いくつかはそれはおそらく何の違い3をなさないと主張それにもかかわらず、優れたRNAを保持する17を与えることになっている0.25％のグルタルアルデヒドを、含まれています。材料を切削後、ハイブリダイゼーションの前に（すなわち、脱蝋再水和し、透過処理、プローブの非特異的結合を減らすために治療し、最後に脱水）スライド上に固定し、前処理されています。 全体の検出手順ここで紹介するプロトコルで、通常の実験室で行うことができる、信号は1〜3日以内に通常の開発と背景上の信号と​​の比率を継続的に監視することができます。 DIG標識プローブは、通常、放射性標識プローブに比べて、より明確なシグナルを与える、より安定しており、連続した実験で再利用することができます。一方、感度は放射性プローブ17に比べて低減されます。situハイブリダイゼーションでは 、特定のmRNAに特異的な標識プローブを使用して式を検出する。ラジオ標識は、堅牢かつ高感度標識法ですが、それは放射能の処理を必要とし、その結果が検出される前にそれは数週間かかる。一方、抗原-標識プローブは、有害性の低い、より安定しており、わずか17数日間検出媒体への露出を必要とする。従って、抗原標識方法は、現在支配している。プロトコルに関係なく最も重要なステップは、プローブの設計です。ケアは、プローブは高い特異性を持ち、高品質のUTPsで標識されていることを確認する注意が必要です。時にはハイブリダイゼーション温度および/または反応の長さは、特定のプローブに調整する必要があります。 DIG標識プローブに基づいて私たちの修正されたプロトコルは、A.に至るまで種で同時にmRNA発現の局在を容易にするP.のシロイヌナズナトドマツ 、およびマイナーの調整で他の植物種で利用できるようになるはずです。プロトコルは、男性のコーンの横に、また植物シュートのさまざまな段階でとPから接合子と体細胞胚の両方でテストされています良い結果（。。カールグレンら未発表結果）とモミ属 。

<table><tbody><tr><td>Acetic anhydride</td><td>Sigma-Aldrich</td><td>A6404-200ml</td><td>minimum 98%</td></tr><tr><td>Acrylamide, linear</td><td>Ambion</td><td>9520</td><td></td></tr><tr><td>Anti-DIG-antibodies</td><td>Roche Group</td><td></td><td></td></tr><tr><td>Blocking reagent</td><td>Roche Group</td><td>11 175 041 910 or 11 096 176 001</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030-50g</td><td>minimum 98%</td></tr><tr><td>Deionized formamide</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Denhardts solution</td><td>Sigma-Aldrich</td><td>D2532-5ml</td><td></td></tr><tr><td>DEPC, diethylpyrocarbonate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Dextran sulfate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>DIG RNA Labeling Kit (SP6/T7)</td><td>Roche Group</td><td>11175025910</td><td></td></tr><tr><td>Glutaraldehyde (25%)</td><td>Histolab</td><td></td><td></td></tr><tr><td>Glycogen</td><td>Ambion</td><td>9510G</td><td></td></tr><tr><td>Histoclear II</td><td>Histolab</td><td></td><td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td></tr><tr><td>Histowax</td><td>Histolab</td><td></td><td>Histowax or Paraplast can be used for embedding.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-500g</td><td></td></tr><tr><td>Paraplast plus</td><td>Sigma-Aldrich</td><td>P3683-1kg</td><td>Histowax or Paraplast can be used for embedding.</td></tr><tr><td>Phusion&amp;#8482; High-Fidility DNA Polymerase</td><td>Finnzymes</td><td></td><td></td></tr><tr><td>Probe-on Plus slides</td><td>Fisher Scientific</td><td>22-230-900</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>P2308-5mg</td><td></td></tr><tr><td>RNase A</td><td>Sigma-Aldrich</td><td>R5503-100mg</td><td></td></tr><tr><td>Tissue-clear</td><td>Sakura Finetek</td><td></td><td>You may use Tissue-clear, Histoclear II, xylen or something similar when embedding your tissue.</td></tr><tr><td>Triethanolamine</td><td>Sigma-Aldrich</td><td>T1377-100ml</td><td>minimum 98%</td></tr><tr><td>Triton X-100</td><td>use your standard product in the lab</td><td></td><td>Use one or both of Triton X-100 and Tween-20.</td></tr><tr><td>tRNA</td><td>Sigma-Aldrich</td><td>83853-100mg</td><td></td></tr><tr><td>Tween-20</td><td>use your standard product in the lab</td><td></td><td>Use one or both of Triton X-100 and Tween-20.</td></tr><tr><td>Western Blue</td><td>Promega Corp.</td><td></td><td></td></tr></tbody></table>

non-radioactive in situ hybridization protocol applicable for norway spruce and a range of plant species

ハイスループット発現解析技術は、現在入手可能なので、個々の組織を解剖時の問題により制限されている科学者に発現プロファイルのオーバーフローが組織特異的発現の観点からその解決方法を与える。発現データは、in situハイブリダイゼーションなどを用いて発現パターンを確認し、補完する必要がある、するために使用される技術は、細胞特異的mRNAの発現をローカライズ。 in situハイブリダイゼーション法 、時間のかかる、面倒であり、しばしば種や組織に応じて大規模な最適化が必要です。 場の実験ではそのような針葉樹ノルウェートウヒ（ ドイツトウヒ ）などの木本種で実行することが比較的困難です。ここでは、P.を含む植物種の広い範囲で高速かつ適用可能なin situハイブリダイゼーションのプロトコールで修正されたDIGを、提示モミ属 。変更されたRNase処理とプロテイナーゼKの濃度を含むだけでいくつかの調整、と、我々一裸子植物二被子植物の種の雄の生殖器官の相同遺伝子の組織特異的発現を研究するためのプロトコルを使用することができます。P.トドマツ、シロイヌナズナとセイヨウアブラナ 。プロトコルは、研究の種との遺伝子にも同様に働いた。AtAP3とBnAP3がAにある2番目と3番目の渦巻き花器官で観察されたシロイヌナズナとB.セイヨウとP.から男性の円錐のmicrosporophyllsのDAL13 モミ属 。 Pの組織をpermeablizeするために使用されるプロテイナーゼKの濃度は、よりコンパクトな組織とフェノール類と多糖類のより高いレベルが原因の可能性ではなく、1〜3 g / mlのg / mlで、に増加する必要があったモミ属 。すべての種のためにRNase処理は、特異性の増加に対応することなく減少し、信号強度のために削除されました。草花や針葉樹の両方から相同遺伝子の組織特異的発現パターンを比較することによって我々は、 その場プロトコルの DIGだけ微調整して、ここで提示した実証は植物種の広い範囲に適用することができます。したがって、プロトコルは広範な生物種固有の最適化とDIG標識プローブを支持して放射性標識プローブの面倒な使用の両方を避けることができます。私たちは、映画におけるプロトコルの技術的な要求の手順を説明するために選択しました。 アンナカールグレンとジェニーカールソンはこの研究にも同様に貢献した。 対応する制作者：Jens.Sundstrom @ vbsg.slu.seでAnna.Karlgren @ ebc.uu.seとJens F.のSundströmでアンナカールグレン

Watch this Scientific Journal Video about Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species at JoVE.com

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

我々は、修正されたDIGを説明<em&gt;その場で</em高速とノルウェートウヒを含む植物種の広い範囲で適用できる&gt;のハイブリダイゼーションプロトコル、。変更されたRNase処理とプロテイナーゼKの濃度を含むだけでいくつかの調整、と、プロトコルは、さまざまな組織や種の研究に使用されることがあります。

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 ...

non-radioactive-situ-hybridization-protocol-applicable-for-norway

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

20.9K ビュー.  Uppsala University. 我々は、修正されたDIGを説明その場でのハイブリダイゼーションプロトコル、。変更されたRNase処理とプロテイナーゼKの濃度を含むだけでいくつかの調整、と、プロトコルは、さまざまな組織や種の研究に使用されることがあります。

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

二重蛍光その場で</em新鮮な脳切片における>ハイブリダイゼーション

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

神経科学

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

その場でパラフィン埋め込まれた大人サンゴの標本の交配の技術

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

環境学

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

蛍光<em&gt;その場での</emウイルスと植物及び昆虫組織における共生細菌の局在化のための&gt;ハイブリダイゼーション（FISH）

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

免疫・感染学

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In Situ ハイブリダイゼーションアステュアナクス胚の Wholemount

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

発生生物学

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

ダブル蛍光を組み合わせます<em&gt;その場で</em&gt;マウス脳切片の3遺伝子の発現を検出するための免疫標識とのハイブリダイゼーション

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

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 ...

生物学

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. ...

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

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

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

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

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization (WMISH) is a common technique used for visualizing the location of expressed RNAs in embryos. In this process, synthetically produced RNA probes are first complementarily bound, or "hybridized," to the transcripts of target genes. Immunohistochemistry or fluorescence is then used to detect these RNA hybrids, revealing spatial and temporal patterns of gene expression. Unlike traditional in situ hybridization techniques, which require thin tissue sections whose images will need to be computationally reassembled, the whole-mount technique allows gene expression patterns to be assessed over the entire embryo or structure.
This video will introduce the basic concepts of whole mount staining and detail key procedural steps, including probe design and production, embryo fixation and staining, and post-hybridization signal detection. Viewers will then learn about how developmental biologists are applying WMISH to current research studies.

非放射性その場でハイブリダイゼーションプロトコール

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二重蛍光その場でハイブリダイゼーション

その場でパラフィン埋め込まれた大人サンゴの標本の交配の技術

蛍光

In Situ ハイブリダイゼーションアステュアナクス胚の Wholemount

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

ダブル蛍光を組み合わせます

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

放射性その場でハイブリダイゼーション

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

ホールマウントIn Situハイブリダイゼーション

非放射性その場でハイブリダイゼーションプロトコール

要約

さらに動画を探す

この動画の章

二重蛍光その場でハイブリダイゼーション

その場でパラフィン埋め込まれた大人サンゴの標本の交配の技術

蛍光

In Situ ハイブリダイゼーションアステュアナクス胚の Wholemount

蛍光による神経組織の持続的なDNAウイルスのゲノム転写物の検出その場でハイブリダイゼーションは、免疫染色と組み合わせる

ダブル蛍光を組み合わせます

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

放射性その場でハイブリダイゼーション

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

ホール マウントIn Situハイブリダイゼーション

ホールマウントIn Situハイブリダイゼーション