この研究の財政的支援は、国家主要研究開発計画（2016YFC1200601）と中国国立自然科学財団（31390421）によって提供された。

1.白蝶貝の飼育と種識別<ol><li> 27±1℃、湿度70±10％、光14時間：暗期10時間の標準条件下で、カゴGossypium hirsutum （Malvaceae）（cv。Zhe-Mian 1973）をカゴで維持する。 </li><li>個々の成熟白コショウを集め、30μLの溶解緩衝液（10mMトリス、pH8.4,50mM KCl、0.45％[wt / vol] Tween-20,0.2％[wt / vol]ゼラチン、0.45％[vol / vol]ノニデットP40,60μg/ mLプロテイナーゼK）。 </li><li>ホモジネートを65℃で1時間インキュベートし、次に100℃で10分間インキュベートする。 注：65℃でのインキュベーション時間は必要に応じて増やすことができます。 100℃でのインキュベーション時間は、Proteinase Kを十分に不活性化し、DNA損傷を避けるために厳密に制御する必要があります。 </li><li> ミトコンドリアシトクロムオキシダーゼIプライマーに基づいて、whitefly DNA（セクション1.3で得られた）を用いてポリメラーゼ連鎖反応（PCR）を行う。 COI-F：5&#39;-TTGATTTTTTGGTCATCCAGAAGT-3 &#39; COI-R：5&#39;-TAATATGGCAGATTAGTGCATTGGA-3 &#39; <ol><li> 1UのTaq DNAポリメラーゼ、10μlのBuffer、0.2mMのdNTP、0.2mMの各プライマー、2μLのwhitefly DNAを含む25μLの最終容量でPCR反応を行います。 </li><li> 95℃で3分間の変性後、95℃で45秒間（変性）、50℃で45秒間（アニーリング）および72℃で1分間（伸長）の35サイクル、およびさらなる伸長のために72℃でさらに10分間。 </li></ol></li><li> DNAゲル抽出キットを使用してPCR増幅産物を推奨プロトコールで洗浄します。 </li><li>精製したDNAサンプルをDNAサンプルシークエンシングシステムで、製造者の指示に従って配列決定する。 </li><li> Basic Local Alignment Search Tool（BLAST）を使用してシークエンスの潜在種を特定し、他の種の汚染を避けるために配列を分析するes。 </li></ol> 2.内分泌攪乱の同定と局在化<ol><li>白蝶状DNA（ステップ1.3で取得）にPCRを行い、白蝶内の各内毒素の特定の遺伝子を増幅する（PCRレシピはセクション1.4に記載されており、PCR手順およびプライマーは表1に記載されている ）。 </li><li>以前に公表されたプロトコール20 （1％アガロースゲル、ランニングバッファーとしてTris-アセテート-EDTA、電圧5V / cm）に従って増幅した試料をアガロースゲル電気泳動に付して、各細菌の特異的遺伝子を同定する。 </li><li>白い葉の中の細菌種を決定するために、各バンドを回収し、配列決定する（1.5-1.7節参照）。 </li><li>以前に公表されたプロトコール19に従う、内腔の位置を同定するために、ウンシュウの蛍光in situハイブリダイゼーション（FISH）を実施する。 注：必要に応じて蛍光プローブの濃度を上げてください。 </li></o1&gt; 透過型電子顕微鏡（TEM） <ol><li> 2.5％[vol / vol]グルタルアルデヒドを含有するリン酸緩衝液（0.1M、pH7.0）中に白鳥を4時間浸漬して固定する。 </li><li>リン酸緩衝液（0.1M、pH7.0）中でサンプルを3回、毎回15分間洗浄する。 </li><li> 1％[wt / vol] OsO 4を含有するリン酸緩衝液（0.1M、pH7.0）に試料を2時間浸して再度固定する。 </li><li>毎回15分の段階的なエタノール（30％、50％、70％、80％、90％、95％および100％）で試料を脱水する。 </li><li>サンプルを100％アセトンに20分間浸して浸します。 </li><li>サンプルを100％アセトンと100％Spurr樹脂（1：1）との混合物中に室温で1時間置く。 </li><li>試料を100％アセトンと100％Spurr樹脂（1：3）の混合物に室温で3時間移す。 </li><li>サンプルを100％Spurr樹脂（70℃でインキュベート）に9時間以上浸して浸します。 </li><li>白を用いて、マイクロを用いてサンプルを切る私に。 </li><li>絶対ウラニルアセテートに5〜10分間浸漬し、続いて50％アルカリ性クエン酸鉛にさらに5〜10分間浸漬することにより、超薄膜（セクション3.9で得たもの）を染色する。 注：セクション3.1-3.10で利用されている試薬の量は、サンプルによって異なります。サンプルが試薬に浸されていることを確認してください。 </li><li>透過型電子顕微鏡（15,000X）でサンプルを観察し、さらなる使用のために細菌の局在、形状およびサイズを決定する（セクション4.6を参照）。 </li></ol> 4.白蝶菌の解剖および精製<ol><li> 1倍PBS溶液100μLを顕微鏡スライドに加える。綿の葉から3〜4齢のニンフをとり、PBS溶液に浸します。 </li><li> 実体顕微鏡下で細かい昆虫の針を使用し、白蝶身からバクテリアを引き出します。 <ol><li>ゆっくりとニンフの片側の穴を切り、もう一方を少し押してくださいバクテリオミセスを排除するための側面。 </li></ol></li><li> 0.5-10μLピペットで20μLのマイクロローダー（細菌のサイズに合うように先端を切断したもの）をマウントする。個々のバクテリオミウムをPBS溶液からマイクロローダーに抽出する。 </li><li>細菌溶液を洗浄して、細菌溶液をPBS溶液にピペッティングし、細菌を抽出することによって、他の白癬組織の汚染を除去する。 3回繰り返します。 </li><li>直ちに、洗浄した細菌を60μLの1×PBS溶液を含む遠心管にピペットで移す。 </li><li>組み立てたバクテリオロームを5μmのフィルター膜（バクテリアのバクテリアと核の大きさで確認）にシリンジでろ過する。混合液をフィルター膜に完全に移動するために複数回繰り返します。 注：増幅とシークエンシングのより良い結果を得るには、100以上の細菌を集めます。細菌の損失を減らすために1x PBS溶液を使用して最初にフィルター膜をぬらしてください膜に結合する。 </li></ol> Endosymbiontゲノムの増幅<ol><li> いくつかの改変を加えた推奨プロトコールによるDNA増幅キットを用いて、ろ液（主にコナジラミの内胞子を含む）を増幅する。 <ol><li>血液または細胞からのゲノムDNAの増幅の推奨プロトコールを選択し、その後の実験のために緩衝液D2を調製する。 </li><li> 1.5μLのろ液（セクション4.6を参照）を遠心チューブに直接添加し、続いて1.5μLのBuffer D2を加えよく混合します。 </li><li>氷上で10分間インキュベートし、停止液1.5μLを加える。 </li><li> 15μLの反応バッファーと1μLのDNAポリメラーゼを添加し、穏やかに混合する。 </li><li>混合物を30℃で16時間インキュベートし、続いて65℃で3分間インキュベートする。 </li></ol></li><li> bac用に設計された特異的プライマーを使用することにより、増幅された濾液上で直接PCRを実施する（必要に応じて増幅されたサンプルを希釈する）細菌種を確認するために使用した（2.1節参照）。 </li><li>以前に公表された方法21 （PCRレシピはセクション1.4に記載されている）に従ってwhitefly遺伝子ベータアクチンおよびEF1プライマーを使用することによって宿主ゲノムの汚染があるかどうかを確認するためにPCRを行う。 </li></ol> 6.エンドサイトーシスメタゲノム配列決定<ol><li>配列の前に増幅されたろ液を分光光度計と蛍光光度計（製造元の指示に従う）に供して、試料の品質および試料が配列決定の基準を満たすかどうかを試験する。 </li><li>増幅したものを製造者の指示に従ってゲノムシーケンサーに供する。 </li></ol>

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A. 89 (7), 2699-2702 (1992).</li><li>Nirgianaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolbachia+infections+of+the+whitefly+Bemisia+tabaci.">Wolbachia infections of the whitefly Bemisia tabaci.</a> Curr. Microbiol. 47 (2), 93-101 (2003).</li><li>Everett, K. D., Thao, M. L., Horn, M., Dyszynski, G. E., Baumann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chlamydiae+in+whiteflies+and+scale+insects:+endosymbionts+'Candidatus+Fritschea+bemisiae'+strain+Falk+and+'Candidatus+Fritschea+eriococci'+strain+Elm.">Novel chlamydiae in whiteflies and scale insects: endosymbionts 'Candidatus Fritschea bemisiae' strain Falk and 'Candidatus Fritschea eriococci' strain Elm.</a> Int. J. Syst. Evol. Micr. 55, 1581-1587 (2005).</li></ol>

著者は何も開示することはない。

Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga...

節足動物1では 、相対的な宿主と密接な共生関係を形成する細菌が広がっている1 。内分泌攪乱物質は、栄養代謝、繁殖、環境ストレスへの応答2,3、4などのような宿主の局面にほとんど全ての発育段階5において影響を及ぼすことが実証されている5 。しかし、協会の基盤となる仕組みはまだほとんど知られていません。ゲノミクスは、細菌の潜在的な機能と役割を研究する際に重要かつ重要です。いくつかの基本的な情報、 すなわち 、分類学的状態、機能遺伝子、代謝経路、分泌系は、共生している共生生物の潜在的役割についての光を放出するゲノム配列から推測することができる。ハイスループット配列決定の開発により、膨大な数の細菌ゲノムが配列決定されている様々な機能を明らかに6 。 内寄生虫は、アブラムシ7 、ナンキンムシ8 、オオバコ9 、褐斑病10および蝉11などの半翅類において極めて重要である。例えば、アブラムシのBuchneraは、必須の共生生物として、アブラムシのゲノム12由来の遺伝子とともに、必須のアミノ酸生合成に関与することが示されている。さらに、 ブフネラ（Buchnera）の転写調節もまた明らかにされている13 。 オオバコでは、 Carsonellaが配列決定され、今までに発見された最小の細菌ゲノムにランクされている14 。これらの内在性の特徴はすべて、ゲノム配列に基づいて推測されます。これらの内在性物質はインビトロで培養することができないので、seqのために適切な細菌を単離するためにいくつかのアプローチが適用されている覚醒する。アブラムシでは、遠心分離および濾過により内腔菌を抽出し、さらなるゲノムおよび転写分析に供する5 。褐斑病菌では、昆虫ゲノム10全体とともに内生菌が配列決定される10 。 Whitefly B. tabaciは、35種以上の形態学的に区別不能な種（潜在種）を含む種複合体であり、そのうち2種の侵略種が世界中に侵入し、農業生産に多大な害をもたらした15 。注目すべきことに、 B. tabaci種内の内在性物質は、害虫16の発生において重要であることを示している。今日まで、8種の内在性物質が、 ハリネリチア 、 リケッチア 、 アルセノフォヌス 、 カルディニウム 、em&gt; Wolbachia、 FritschaおよびHemipteriphilusは、17,18を定義した。 以前に記載された半体とは異なり、コナジラミB. tabaciはわずか1mmの非常に小さな昆虫である。ほとんどの内在性物質は、バクテリア細胞19 （B細胞において細菌をさらに形成する共生物質を含む特殊細胞）に限定される。さらに、これらの内在性物質はインビトロで培養することができない。バチルス・タバチ（ B. tabaci）から内寄生虫を得るための唯一の方法は、細菌を解剖することである。しかし、解剖が困難である。第一に、脆弱な細菌は、常に分離するのは難しい白蝶の他の組織とつながります。第二に、白蝶の小さなサイズは、十分な細菌の分離を制限する。第3に、内在性細菌は細菌の中に集まり、単一の細菌種を獲得することを非常に複雑にする。

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Taq DNA polymerase</td>
			<td style="width:107px;">Takara</td>
			<td style="width:129px;">R001A</td>
			<td style="width:296px;">including rTaq, 10&#215;Buffer and dNTP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Gel DNA extraction kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">28704</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">DNA sample sequencing system</td>
			<td style="width:107px;">ABI</td>
			<td style="width:129px;">ABI-3730XL</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Microtome</td>
			<td style="width:107px;">Leica</td>
			<td style="width:129px;">EM UC7</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Transmission electron microscopy</td>
			<td style="width:107px;">Hitachi</td>
			<td style="width:129px;">H-7650 TEM</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Stereo microscope</td>
			<td style="width:107px;">Zeiss</td>
			<td style="width:129px;">Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">20 &#956;L microloader</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:129px;">F2771951</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter holder</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SX0001300</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter membrane filter</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SMWP001300</td>
			<td>5.0 &#956;m SMWP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">REPLI-g UltraFast Mini Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">150033</td>
			<td>DNA amlification kit</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">NanoDrop</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:129px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Qubit Fluorometer</td>
			<td style="width:107px;">Thermo Fisher Scientific</td>
			<td style="width:129px;">Q33216</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Genome Sequencer</td>
			<td style="width:107px;">Illumina</td>
			<td style="width:129px;">Hiseq 2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for the extraction of endosymbionts from the whitefly bemisia tabaci

細菌共生生物は、宿主と親密な関係を形成し、ほとんどの場合宿主に利点を与える。ゲノム情報は、宿主における細菌共生生物の機能および進化を研究するために重要である。ほとんどの共生物質はインビトロで培養できないため、ゲノム配列決定のために適切な量の細菌を単離する方法は非常に重要です。コナジラミBemisia tabaciでは、いくつかの内寄生虫が同定されており、複数のアプローチによる有害生物の発生および生殖において重要であると予測されている。しかし、協会を支える仕組みはほとんど知られていません。この障害は部分的に、白癬菌の内在性物質（ほとんどが細菌細胞に抑制されている）が宿主細胞から分離するのが困難であるという事実に由来する。ここでは、主に解剖学的検査によって白癬B. tabaciからの内在性物質の同定、抽出および精製のための段階的プロトコールを報告するオンにして濾過する。この方法によって調製された内毒素試料は、依然として異なる内毒素種の混合物であるが、その後のゲノム配列決定およびバチルス・タバチにおける内在性の可能な役割の分析に適している。この方法は、他の昆虫から内生菌を単離するためにも使用することができる。

ここでは、 B. tabaci複合体の中東アジアマイナー1（MEAM1）種を例として取り上げた。コナジラミを飼育するためのコットンおよびコナジラミのいくつかの発生段階を、 図1に示す。コットン植物、成虫コナジラミ、およびコナジラミの第1、 第 2 および第 4齢の幼虫（第3齢の幼虫は第 4齢の幼虫と同様に見える）。第...

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

ここでは、白斑Bemisia tabaciからの内胚葉を切開および濾過により単離するためのプロトコールを提示する。増幅後、DNAサンプルは、その後のシークエンシングおよび内寄生虫とコナジラミ間の共生の研究に適している。

白蝶貝からの内生菌の抽出方法<em&gt;ベミシア・タバチ</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

methods-for-extraction-endosymbionts-from-whitefly-bemisia

Research

JoVE Journal

Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

9.8K ビュー.  Zhejiang University. ここでは、白斑Bemisia tabaciからの内胚葉を切開および濾過により単離するためのプロトコールを提示する。増幅後、DNAサンプルは、その後のシークエンシングおよび内寄生虫とコナジラミ間の共生の研究に適している。 

ビデオ: Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

がくからパッチクランプの静電容量の録音のための方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

生物学

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

RNAまたは特定の脳領域からのタンパク質抽出用のラット脳の微小解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Bio - Rad社のTotal RNA Kitを使​​用してNeuroprecursor細胞からのRNA抽出

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

ショウジョウバエの幼虫のNMJからシナプス電位を記録するための電気生理学的方法

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

ゼブラフィッシュ上顎バーベルの研究のための方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Extraction of the EPP Component from the Surface EMG

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.

The endplate potential (EPP) component can be extracted from the surface EMG using a digital filter. The extracted EPP shows oscillation with a frequency of about 30 Hz.

表面筋電からEPPコンポーネントの抽出

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

植物の葉からのアポプラスト液の抽出のための浸透·遠心分離技術の使用<em&gt;インゲンマメ</em&gt;例として

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

成体マウスから洞房結節筋細胞の単離、培養、および機能解析のための方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...