Ghanimラボでの研究は研究助成金なしでサポートされていました。ドイツ·イスラエル財団（GIF）から908-42.12/2006は、無許可する。 IS-4062から07米国 - イスラエル二国間農業研究と開発基金（BARD）、および研究助成金がないから。 MGにイスラエル科学財団（ISF）から884/07

1。 FISH解析のための一般的なコナジラミ、プラント、およびウイルス調製<ol><li>リアBとQバイオタイプ綿の苗（ ワタヒルスツム L.品種不動明王）にコナジラミおよび25±2℃、相対湿度60％の標準的な条件下で防虫ケージと成長室の内部で維持し、14時間の明/ 10時間暗の光。 </li><li>前に説明した15〜19のように細胞内共生特異的プライマーを用いて内部共生の感染をテストするためにPCRを行う。 </li><li>商用保育園や植物トマトの種子からトマトの苗（ ナスエスクレンタムビーフステーキ）を購入。 </li><li>植物はTYLCV 15のコナジラミ媒介接種に最も適した年齢は（セクションの下に4.1を参照）、4真葉期に達するまで、上記と同様の飼育条件で維持する。 </li></ol> 2。遠藤昆虫処理、固定、プローブ設計、ハイブリダイゼーション、および可視化共生FISH <ol><li>綿植物からの吸引により10大人のコナジラミを収集、または綿の葉から10 第 3または第 4齢幼虫を選ぶ。 </li><li>すぐにエッペンドルフチュー​​ブ内部のカルノア固定液（クロロホルム - エタノール - 氷酢酸、6時03分01秒、体積/体積）に浸す。一晩に2時間（好ましくは一晩）を修正しました。 </li><li>完全に固定液を除去し、2時間、エタノール中の6％H 2 O 2で脱色。 </li><li>室温で数日から数週間のための無水エタノール中のサンプルを保存したり、次のステップに進みます。 </li><li>各細菌の16SリボソームRNA遺伝子に基づいて補完的なリバースDNAプライマーとして設計プローブ。プローブを設計するために、考慮にPCRプライマーを設計するための同じ考慮がかかる。オリゴヌクレオチドプローブを使用BTP1 5&#39;-Cy5でTGTCAGTGTCAGCCCAGAAG-3 &#39;、5&#39;-Rb1をCy3でTCCACGTCGCCGTCTTGC-3&#39;、5&#39;-BTHのCy3-CCAGATTCCCAGACTTTACTCA-3 &#39;および5&#39;-Ars2のcy3-TCATGACCACAACCTCCAAA-3 &#39;は、それぞれPortiera、リケッチア、Hamilonella、およびArsenophonus、の特異的標的化のために。 </li><li> ml当たり10pmolの蛍光プローブを含有する暗ハイブリダイゼーション緩衝液中（20mMのトリス-HCl、pH8.0、0.9 MのNaCl、0.01％[重量/容量]ドデシル硫酸ナトリウム、30％[容量/容量]ホルムアミド）中で一晩サンプルをハイブリ。必要に応じて、異なる蛍光色素で、複数のプローブを兼ね備えています。ネガティブコントロールとして目標と細菌と非プローブのサンプルと非感染コナジラミを使用してください。 </li><li>液体ブロッカーを備えた自給ハイブリダイゼーション緩衝液中で、顕微鏡スライド上に、全体のサンプルをマウントカバーガラスで覆い、蛍光または共焦点顕微鏡下でのマニキュア、およびビューで密封する。 </li></ol> 3。コナジラミオルガン解離、ボール、ハイブリダイゼーション、およびTYLCVの魚のための可視化<ol><li>連続して吸引とANEでTYLCV感染トマト植物で飼育大人のコナジラミを収集アセトンを含浸させた紙タオルに暴露することによってsthetize。アセトン長すぎて露出がコナジラミ組織を固定できるようにわずか1〜2分間コナジラミを公開します。特異的ハイブリダイゼーションを確認するために、非感染トマト植物とコントロールをなしプローブサンプルで飼育nonviruliferousコナジラミを使用してください。 </li><li>うつ病顕微鏡のマウントコナジラミは、実体顕微鏡を用いて、臓器の解剖のためにスライドさせる。 </li><li>前胸から昆虫ヘッドを引き離すと、1X PBS中に唾液腺を解剖は、その小さなサイズのため、より良い視覚化のために、1％トルイジンブルー染色を補った。 2〜3分間汚れの吸収を可能にします。 </li><li>観察し、腸を排出するために胸部と腹部の間の接合部で離れて昆虫を引く。腹部先端に鉗子で押すことで、腹部の内容を追放。 </li><li>静かに成功した解剖からPBSおよびトルイジンブルーを削除して、ゆっくりと5分間の臓器を固定するためのカルノア固定液を300μlを加える。 </li><li> A解剖した臓器の制御不能な動きを防止するために落ち込んウェルの片側からではなく、上から固定液をD-D固定液に触れると、臓器がガラスに付着し、プロセス全体を通じて、彼らは移動しません。 </li><li>固定液を除去してTYLCVコートタンパク質遺伝子の配列に相補的な蛍光DNAプローブのCy5-5&#39;-GGAACATCAGGGCTTCGATA-3 &#39;、10pmolの補充したハイブリダイゼーション緩衝液500μlを加える。 </li><li>下部に濡れタオルペーパーで小さなプラスチックの箱で構成小さな加湿チャンバー内部の試料をスライドをインキュベートすることにより、暗所で室温で一晩ハイブリダイズ。 </li><li>ハイブリダイゼーション後、細かい解剖ツールで臓器をピックアップし、急速にDAPI（1X PBS中0.1 mg / ml）を補充した液体ブロッカーに含まれている新たなハイブリダイゼーション緩衝液30μlを含有する新鮮な顕微鏡スライドに移動します。 </li><li>カバーガラスで標本をカバーする、ウィットをシール蛍光または共焦点顕微鏡下でのHのマニキュアとビュー。 </li></ol> 4。 TYLCVの魚のための植物の処理、ハンド切片、固定、及びハイブリダイゼーション<ol><li>病原体を持つコナジラミ媒介接種を使っTYLCVとトマトの苗に接種。ウイルスは、無症候性植物におけるこのFISH法接種後一週間を用いて検出することができる。典型的な病気の症状は、目に見える3週間後接種である。特異的ハイブリダイゼーションを確認するためのコントロールの非感染トマト植物なしプローブのサンプルを使用してください。 </li><li>トマトのハンドカット2〜4センチメートル長い縦または断面を製造するための鋭利な組織学的かみそりの刃を使用して、茎葉。 </li><li>エッペンドルフチュー​​ブ中で、2時間まで、室温で一晩のためにカルノア固定液中でのセクションを修正。 </li><li>固定液を除去して、室温で数日から数週間のために無水エタノールにセクションを保存したり、次のステップに進みます。 </li><li>セクションを洗うハイブリダイゼーション緩衝液中で3回、各々1分、。 </li><li> TYLCVコートタンパク質遺伝子内の配列に相補的な蛍光性オリゴヌクレオチドプローブのCy3-5&#39;-GGAACATCAGGGCTTCGATA-3 &#39;10pmolのを補充したハイブリダイゼーション緩衝液500μlで切片をハイブリダイズする。 </li><li>ハイブリダイゼーション緩衝液中で、各項を3回、1分ごとに洗浄します。 </li><li> DAPI（1×PBS中0.1 mg / ml）を補充した液体ブロッカーに含まれているハイブリダイゼーション緩衝液中のホールマウント部分は、蛍光または共焦点顕微鏡下でのマニキュアとビューで密封、カバーガラスでカバーしています。 </li></ol>

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著者らは、開示することは何もありません。

その具体的なコナジラミホストでの植物宿主と昆虫ベクターにおける植物ウイルスの局在のためにここで説明するプロトコル、および共生細菌は、植物であっても動物組織内の他のウイルスの局在に適応させることができる。さらに、プロトコルは、植物および動物系において共生および病原性細菌および他の微生物を局在化するために使用することができる。記載された方法は、短い、蛍?...

それらの感染植物宿主と共に植物ウイルスおよび他の病原体の間の相互作用を研究するとき、それらはそれらの宿主に悪影響を引き起こすかどうかにかかわらず、 その場での病原体及びそれらのそれぞれの核酸を可視化することが重要である。植物細胞内病原体との間の動きを研究する場合に最も重要である。病原体の遺伝子産物のin situ局在化において病原性プロセスを研究するための他のアプローチを補完する重要なステップである。多くの植物病原体、特にウイルスは、それらのベクトルとの複合体との親密な相互作用を持つ、昆虫によって送信される。そのベクターでは、これらのウイルスの局在は、伝送のための経路、およびベクトル内部の可能な相互作用部位を研究するために重要である。いくつかの昆虫ベクトル化植物ウイルスの送信は、これらの昆虫内に存在する共生細菌によって助けられる。よりよいこれらの植物ウイルスBの送信を研究するために、yは、それらのベクターは、それは、特に、一般的な共生細菌およびウイルス伝播に関与するものを視覚化することも重要である。ウイルスや共生細菌の共局在は、このように彼らの昆虫宿主内でこれらの生物間の可能性のある関係を調査のために望ましい。脇にウイルスを送信してから、共生細菌が昆虫のベクターの生物学のいくつかの側面に影響を与え、昆虫内でのこれらの細菌のこのような空間局在は、高い関心と重要である。 トマト黄化葉巻ウイルス （TYLCV）（ベゴモウイルス、Geminiviridae）は、世界中で栽培トマト1-4の中で最も重要なウイルス性疾患の複合体である。 TYLCVは師部限定のウイルスであり、独占的にコナジラミコナジラミコナジラミ 5,6によってベクトル化されている。そのコナジラミのベクターにbegomovirusesの移行を記述するモデルは6-9提案されている。二つの具体的な障壁は、積極的にDUR交差している腸/血リンパおよび血リンパ/唾液腺の障害：この循環送信をING。この送信処理は、ウイルスキャプシドを認識し、未知の受容体によって媒介されると仮定される。 TYLCVはBを横断すると考えられているコナジラミの腸6,10、それが植物内に注入する前に、プライマリ唾液腺6を介して吸収される。コナジラミ血リンパでは、TYLCVは昆虫の二次共生細菌Hamiltonellaによって生成のGroELタンパク質と相互作用する。この相互作用は、血リンパにTYLCVの安全な輸送を確保し、昆虫の免疫系11。HamiltonellaとPortiera、一次細胞内共生による攻撃から保護するコナジラミの、bacteriocytes、血リンパやハウス内共生細菌11で見つかった昆虫細胞内に収容されている。B.コナジラミはリケッチア、Arsenophonus、ボルバキアとフリッチュ含む追加の共生細菌を保有bacteriocytes内側または外側に局所化することができ、EAは、昆虫の生物学12に多様な効果を持っている。 いくつかの報告が微小厚部10,13上にその場で 、例えば透過型電子顕微鏡（TEM）、抗体およびRNAのような時間がかかり、高価なプロトコルを用いて植物およびコナジラミにおけるTYLCVの局在化を研究するために試みられているが、最近の研究では、局在化を記載単純なプロトコル14を使用して、彼らの工場とベクトルのホスト内部の植物ウイルスの。ここでは、BのTYLCVの局在化のための単純なプロトコルを記述タバココナジラミは、中腸と唾液腺を解剖して、セクションでTYLCVに感染した植物から調製した。我々はさらにPortiera、Bの一次細胞内共生の局在を説明コナジラミ、およびその二次共生Hamitonella、リケッチアおよびArsenophonus。このプロトコルは、短いDNAプローブを用いることに基づくということです蛍光5 &#39;末端に標識され、具体的には、ウイルスまたは細菌の遺伝子配列中の相補配列にハイブリダイズされる。サンプル処理は比較的容易であり、得られた信号は、非常に特異的である。記載されたプロトコルは、それらの植物、動物および昆虫宿主においてウイルス、細菌および他の病原体を局在化するために使用することができ、さらに、任意の所与の組織中のmRNAを局在化するために使用することができる。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fluorescently labeled&#160; Probes</td>
			<td style="width:95px;">Metabion</td>
			<td style="width:129px;">20 bp HPLC purified</td>
			<td style="width:212px;">Sequence designed by customer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Toluidine blue</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">89640</td>
			<td style="width:212px;"></td>
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			<td height="21" style="height:21px;width:219px;">sodium dodecyl sulfate</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">L3771</td>
			<td style="width:212px;">Molecular Biology Grade</td>
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			<td height="21" style="height:21px;width:219px;">Formamide</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">F9037</td>
			<td style="width:212px;">Molecular Biology Grade</td>
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			<td height="21" style="height:21px;width:219px;">Tris-HCl</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">T5941</td>
			<td style="width:212px;">Molecular Biology Grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Glacial Acetic Acid</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td style="width:129px;">320099</td>
			<td style="width:212px;">Molecular Biology Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Blocker</td>
			<td>Ted Pella Inc.</td>
			<td>22309</td>
			<td></td>
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fluorescence in situ hybridizations (fish) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues

in situハイブリダイゼーション （FISH） の蛍光は、一般に、真核細胞における遺伝子転写産物を可視化し、さらに、ウイルスや細菌による感染などの細胞内の他のコンポーネントを視覚化するために修飾することができるために使用される技術の様々に与えられた名称である。空間的局在および感染プロセス中のウイルスや細菌の可視化は、様々な刺激に応答してそのようなマイクロアレイおよびRNAseqとして発現プロファイリング実験を補完する重要なステップです。これらの試薬を用いた時空間の感染症を理解することは、細胞成分との相互作用を理解することを目的とした生物学的な実験を補完するものです。このようなレポーター遺伝子システムまたは免疫組織化学的方法などのウイルスおよび細菌を視覚化するためのいくつかの技術は時間がかかり、そしていくつかはモデル生物で動作し、複雑な方法論を含むよう限定される。細胞内のRNAまたはDNA種を対象としたFISHは比較的容易で、速い私です遺伝子の時空間局在を研究するための診断目的のためにTHOD。この方法は、堅牢でプロトコルは高価ではありません短いダイズ、商業的に購入したプローブを用いた場合に実施するのが比較的簡単にすることができます。試料調製、固定、ハイブリダイゼーション、および顕微鏡的視覚化は、複雑な工程を含まない場合に特に強固である。ここでは、昆虫や植物組織中の細菌やウイルスの局在化のためのプロトコルを記述します。この方法は、その5 &#39;または3&#39;末端に蛍光色素に結合させ20塩基対の短いDNAプローブを用いた簡単な準備、固定、および昆虫ホールマウントし、解剖し臓器や手作りの植物切片のハイブリダイゼーションに基づいています。このプロトコルは、正常昆虫および植物組織の数に適用されており、細胞内のmRNAもしくは他のRNAまたはDNA種の発現を分析するために使用することができる。

この原稿で研究システムは、 図1に示されており、TYLCVに感染した植物は、成人およびコナジラミBのニンフが含まれているコナジラミ、昆虫におけるTYLCV転のための経路を示すコナジラミの内部解剖図2は 、一次共生生物Portieraと二次共生生物Arsenophonusのための大人のコナジラミに二重魚を示しています。図3は、Portiera</str...

Watch this Scientific Journal Video about Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues at JoVE.com

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

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

ここではin situハイブリダイゼーションの単純な蛍光（FISH）昆虫や植物組織中のウイルスや細菌の局在のための方法を説明します。このプロトコルは、全体のマウントおよび顕微鏡用切片におけるmRNAの可視化のために拡張することができます。

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 ...

fluorescence-situ-hybridizations-fish-for-localization-viruses

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

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Viruses

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23.2K ビュー.  Volcani Center. ここではin situハイブリダイゼーションの単純な蛍光（FISH）昆虫や植物組織中のウイルスや細菌の局在のための方法を説明します。このプロトコルは、全体のマウントおよび顕微鏡用切片におけるmRNAの可視化のために拡張することができます。 

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

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

ロックされた核酸のフローサイトメトリー、蛍光その場でハイブリダイゼーション（LNAフロー- FISH）：細菌のsmall RNAの検出のための方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

免疫・感染学

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

哺乳動物細胞に関連して細菌の生存を決定するための蛍光顕微鏡法

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

キメラマウスモデルにおける組換えインフルエンザワクチンの鼻腔内投与は、粘膜免疫を研究するために、

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

16S rRNAのをターゲットジゴキシン標識DNAプローブを用いたパラフィン包埋組織内細菌のその場検出で

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

コットンボールワームでデンソウイルスのホストフィットネス上のホスト組織分布、伝送モード、および効果を調査するためのプロトコル

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

蛍光ベースのノイラミニダーゼ阻害アッセイ抗ウイルスのノイラミニダーゼ阻害剤クラスにインフルエンザウイルスの感受性を評価します

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染細胞における特定遺伝子産物の高量蛍光ハイブリダイゼーション(FISH)と免疫蛍光(IF)における定量蛍光

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Localization and Quantification of Begomoviruses in Whitefly Tissues by Immunofluorescence and Quantitative PCR

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. Considering the extensive damage caused by begomoviruses to crop production worldwide, it is imperative to understand the interaction between begomoviruses and their whitefly vector. To do so, localization and quantification of the virus in the vector tissues is crucial. Here, using tomato yellow leaf curl virus (TYLCV) as an example, we describe a detailed protocol to localize begomoviruses in whitefly midguts, primary salivary glands, and ovaries by immunofluorescence. The method is based on the use of specific antibodies against a virus coat protein, dye-labeled secondary antibodies, and a confocal microscope. The protocol can also be used to colocalize begomoviral and whitefly proteins. We further describe a protocol for the quantification of TYLCV in whitefly midguts, primary salivary glands, hemolymph, and ovaries by quantitative PCR (qPCR). Using primers specifically designed for TYLCV, the protocols for quantification allow the comparison of the amount of TYLCV in different tissues of the whitefly. The described protocol is potentially useful for the quantification of begomoviruses in the body of a whitefly and a virus-infected plant. These protocols can be used to analyze the circulation pathway of begomoviruses in the whitefly or as a complement to other methods to study whitefly-begomovirus interactions.

We describe an immunofluorescence and quantitative PCR method for the localization and quantification of begomoviruses in insect tissues. The immunofluorescence protocol can be used to colocalize viral and vector proteins. The quantitative PCR protocol can be extended to quantify viruses in whole whitefly bodies and virus-infected plants.

ホワイトフライ組織におけるベゴモウイルスの免疫蛍光および定量PCRによる局在化と定量化

Begomoviruses (genus Begomovirus, family Geminiviridae) are transmitted by whiteflies of the Bemisia tabaci complex in a persistent, circulative manner. ...