私たちは、ハエの株式を提供するためのブルーミントンショウジョウバエストックセンターに感謝します。我々はまた、抗体を提供するための発達研究ハイブリドーマバンク博士リン·クーリー（エール大学）を感謝。特別な感謝は、原稿上で彼女の助けをAhHyunユに行く。この作品は、自然科学とカナダの工学研究評議会（クリーガー）、ウィリアムとエイダイザベルスチール基金（クリーガー）、およびカナダ衛生研究所（ハーデン）からの補助金によってサポートされていました。

1.ボディウォール準備 NOTE：（体壁の筋肉を神経支配のNMJの研究のための）第三齢幼虫の体壁の調製先にブレントに記載したように行ったら 10、またはラマチャンドラとBudnik 11,12が、いくつかの修正を加えた。 <ol><li>解剖<ol><li>標準的な手順13を使用して五から六日間、25℃でフライ株式や十字架を上げる。 </li><li>細かい鉗子を使用してバイアルまたはボトルから3齢幼虫をクロールピック。 </li><li>任意の食物粒子を除去するためにリン酸緩衝生理食塩水（PBS）を含有する小さなペトリ皿に幼虫を洗浄する。 </li><li>シルガードディスクに、単一の幼虫を置き、氷冷PBSの数滴でそれを浸す。氷冷したPBSを使用すると、簡単に操作すること幼虫を気絶させるのに役立ちます。解剖を通して、準備は常に乾燥からそれを防ぐために、PBS中に沈められていることを確認してください。 </li><li>ラを配置します2気管路は解剖顕微鏡（ 図2A）の下に表示されるようにその背側を上にしてRVA。鉗子を使用して、minutienピンを把持気門（ 図2B）の近くに後端で幼虫を突き止めるする。別のピンを使用すると、口のフックに近い前方の終わりにキューティクルを貫通。ゆっくりと（ 図2B）、それを突き止めるその後、縦に幼虫を伸ばす。 </li><li>顕微解剖はさみを使って、小さな開口部を作成するために、ピンの近くに後部の端をつまんで。切開はちょうどキューティクルを通過するのに十分表面的でなければならない。 </li><li> 2気管路（ 図2C）との間の背側正中線の全長に沿って切断し、切開部に鋏の底刃の先端を配置する。腹側体壁の筋肉を損傷しないように切断する際に、わずかに上向きにはさみの刃を向け。 </li><li>小さな水平切開を行い、わずかにポストの前方eriorly-置かピン（ 図2C）。別の類似の切開がやや前方に-置かピン（ 図2C）に後方を確認します。切開はちょうどキューティクルを通過する必要があります。 注：似ている必要があります合わせてステップ1.1.7と1.1.8から3切開」 <img fo:content-width="0.09in" src="/files/ftp_upload/52139/52139_letter_I.jpg" width="15px" />完成した時」、 すなわち 、幼虫の体の背側の左右のフラップが生成されるべきである。 </li><li>慎重にピンセットで内臓をきれいに。 PBSの少数の力強い滴を追加すると、幼虫の体から臓器を変位ため、それが簡単にそれらを削除することに役立ちます。それは体壁の筋肉の損傷の原因になりますように幼虫の体を突っついは避けてください。 </li><li>オープン幼虫の体を広げると（ 図2D）のコーナーを突き止める。ピン止めするとき、均等に張力をかけ長方形を形成するように水平方向と垂直方向の両方で体壁を伸ばす（それ形状の電子図2G）、その過程で体壁の筋肉を引き裂くないように注意しながら。 </li><li>残りの内臓（ 図2E）を除去完了。 </li></ol></li><li>固定および透過<ol><li>ブアン溶液を数滴に固定体の壁を浸し。氷上で15分間インキュベートする。あるいは、代替固定液として4％パラホルムアルデヒド（PFA）を使用。 30分間インキュベートする。 </li><li>トリトンとのリン酸緩衝生理食塩水（PBT）で3回洗浄します。 </li><li>細かい鉗子を使用して、慎重にピンを取り外し、シリコン処理0.65ミリリットルマイクロ遠心チューブに彼らのコーナーによって身体の壁を転送する。 </li><li> PLAの準備が整うまで4℃でPBTの体壁を保管してください。最適な結果を得るために、一日または解剖の2以内に身体壁を免疫染色始める。 注：試薬上のアイコンをクリックすると、すべての体壁がアッセイ中に平等に扱われることを保証するために、異なる遺伝子型は、単一の中に入れることができるチューブ。遺伝子型（例については、図2Fを参照）異なっ体壁の角を切断することによって区別することができる。 </li></ol></li></ol> 2.免疫組織化学注：三齢幼虫の体壁の免疫染色は、以前にブレントら 14とラマチャンドランとBudnik 11に記載のように行ったが、いくつかの変更11,14とした。 注：特に明記しない限り、室温で、穏やかに攪拌しながら、すべての手順を実行します。 <ol><li>阻止<ol><li> 10分間ずつPBTで三回体の壁を洗ってください。 </li><li> 1時間、1％ウシ血清アルブミン（BSA）でブロックする。 </li></ol></li><li>免疫染色<ol><li>目的の2つのタンパク質に対するマウスおよびウサギ一次抗体と体壁をインキュベートし、室温で2時間、または4℃で一晩のために（1％BSAで希釈）。 250ウサギaの場合：この場合は、1：10のマウス抗は、Dlgおよび1を使用NTI-HTSM 15,16。マウスまたはウサギで行われていないマーカーに対する抗体も含まれることができる- 例えば 、1を使用：200ヤギ抗HRPニューロン膜を描写する。 </li><li> PBTで10分間ずつ3回洗浄する。 </li><li>室温で2時間、または4℃で一晩のために（1％BSAで希釈）のマーカーを検出するためのフルオロフォア結合二次抗体と共にインキュベートする。ヤギ抗HRP抗体を検出するために、200 FITC結合抗ヤギ： 例えば 1を使用する- PLA信号は後で赤色蛍光体で可視化されるように、別のフルオロフォアは、マーカーを検出するために使用されなければならない。による感光性試薬の使用、以降この点から、暗所で管を保持する。 注：PLAは、共焦点顕微鏡下で赤チャンネル上で可視化信号と、マウスおよびウサギで育った一次抗体との間で実行されるために使用されているキットができます。所望であれば、他のキットは、アッセイがプリマで行うことができるように利用可能であるRY他の種で作製された抗体、および他のチャネル上で可視化シグナル。 </li></ol></li></ol> 3.近接ライゲーションアッセイ注：特に明記しない限り、室温で、穏やかに攪拌しながら、すべての手順を実行します。 <ol><li> PLAプローブ<ol><li> 10分間ずつPBTで三回体の壁を洗ってください。 </li><li> 37℃で2時間：（1％BSAで5希釈の各1）PLAプローブとインキュベートする。この場合、5-10体壁の適切な浸漬及び混合を確実にするためにPLAプローブ抗マウスMINUS40μlの、PLAプローブ抗ウサギPLUS40μlの1％BSAの120μlのを使用する。 PLAプローブ1:25希釈までまだ（この実験のために、すなわち 、）十分な信号対雑音比をもたらすことができる。 </li></ol></li><li>ライゲーション<ol><li> 5分間ずつ二回洗浄バッファーAで体の壁を洗ってください。 </li><li> 1時間aのライゲーション溶液（連結緩衝液中でリガーゼの1：40希釈）でインキュベートT 37℃。この場合は、5〜10体の壁の適切な浸漬及び混合を確実にするためにリガーゼの5μL、5ライゲーション緩衝液40μlの高純度水を155μlのを使用しています。 </li></ol></li><li>増幅<ol><li> 2分間ずつ二回洗浄バッファーAで体の壁を洗ってください。 </li><li> 37℃で2時間、増幅溶液（増幅緩衝液中のポリメラーゼの1:80希釈）と共にインキュベートする。この場合は、5〜10体の壁の適切な浸漬及び混合を確実にするためにポリメラーゼ2.5μlの、5増幅緩衝液40μlの高純度水の157.5μLを使用しています。 </li></ol></li><li>イメージングのための準備<ol><li> 10分間ずつ二回洗浄緩衝液Bで体の壁を洗ってください。 </li><li> 1分間に一度0.01X洗浄緩衝液Bで洗浄する。 </li><li>取り付けの前に少なくとも30分間取付溶液数滴で平衡化、または4℃で一晩保存する。 </li><li>微細なピンセットを使用して、慎重にプラットフォームスライドワットに体壁を転送するi番目の彼らのキューティクルを下に向けて。行と封入の低下や2内の同じ向きで体壁を配置します。その後明確なマニキュア液でスライドをシール、気泡を発生させないように注意しながら準備を介して22ミリメートル×40 mmのカバースリップを置きます。 </li><li>共焦点イメージングのための準備ができるまで-20℃の暗所でスライドを保管してください。 </li></ol></li></ol>

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著者らは、開示することは何もない。

このレポートは、PLAは、ショウジョウバエの幼虫NMJに適用する方法を示しています。アッセイは、NMJで存在する内因性のタンパク質 - タンパク質相互作用を検出するため幼虫体壁筋調製物に対して行われる。この技術では、は、Dlgおよびフーバーは、互いに近接しているため、特にシナプス後領域27において、複合体中に存在することが示されている。この結果を支持して、以...

（Dlgという）大ショウジョウバエディスクは、原形質膜の特定の部位での大きなタンパク質複合体の集合を編成助ける足場タンパク質の膜結合グアニル酸キナーゼファミリーの保存されたメンバーである。腫瘍抑制タンパク質は、Dlgという上皮apicobasal極性1,2,3の重要な決定因子として機能するように最初に同定された。 DLGはまた幼虫の発育4中のグルタミン酸作動性運動ニューロンの神経筋接合部（NMJ）での主要な足場モジュールとして提供しています。 DLGは幼虫NMJでの多様な役割を果たし、その多面発現は、複数のタンパク質5,6と会合する能力に依存しています。このようなタンパク質の1つは、胡-LIの太極拳シャオ（HTS）、主にアクチンスペクトリン細胞骨格7の調節における役割に関してに記載されている哺乳類adducinsにホモログである。これは、以前には、Dlgおよびフーバー、インビトロに基づいて、互いに複合体を形成することができることが示されている</em&gt;の全体の大人のフライ溶解物8を含む共免疫沈降実験。これらの結果の一つの欠点は、しかし、それらはここで、この複合体の形態を示すものではないということである。免疫組織化学を使用することにより、Dlgというおよびフーバーの分布は、幼虫のNMJのシナプス後膜にオーバーラップすることが観察されますが、この領域8で複合体中にありますか？最近示されており、ここでさらに詳述されるように、近接ライゲーションアッセイ（PLA）は、幼虫のNMJ 27で特異的には、Dlgとフーバーの間にその場での関連で検索するために使用されます。 PLAは、主にその場 9 でのタンパク質-タンパク質相互作用を検出することができ、細胞および組織培養に使用される比較的新しい技術である。このアッセイでは、目的の2つのタンパク質に対する一次抗体は、種特異的二次抗体の対を用いて検出される、オリゴヌクレオチドにコンジュゲートされたPLAプローブと呼ばれる（ 図1A、B）。 2タンパク質の場合sは、添付のPLAのプローブ間の距離は、2つの追加のコネクターオリゴヌクレオチド（ 図1C）のハイブリダイゼーションを介してブリッジすることができる（数十ナノメートル以内すなわち ）は、互いに近接している。この立体配座において、コネクタオリゴヌクレオチドの自由端が互いに接触するのに十分近く、閉じた環状DNA分子は、in situ ライゲーション （ 図1D）に基づいて形成することができる。環状DNA分子は、PLAプローブ（ 図1E）にコンジュゲートしたオリゴヌクレオチドのいずれかによって準備され、その場でのローリングサークル増幅のための鋳型として働く。得られた増幅、コンカテマーDNA産物内の配列は、次いで、蛍光標識された相補的オリゴヌクレオチドプローブ（ 図1F）で可視化することができる。増幅されたDNAは、PLAプローブの1つは、タンパク質 - タンパク質相互作用withiの細胞内局在に付着したままであるので組織ナ容易に確認することができる。 いくつかの方法は、一般に、共免疫沈降、プルダウンアッセイおよび酵母ツーハイブリッドスクリーニングなどのインビトロ技術において 、そのようなフェルスター共鳴エネルギー移動（FRET）と二分子蛍光相補性（のようなインビボ技術を含むタンパク質-タンパク質相互作用を検出するために使用されるBIFC）。 インビトロ技術の落とし穴は、相互作用が内生的に発生している場所で 、前述のインビボ技術は、それらの内因性の対応のネイティブ動作を反映していない可能性が融合タンパク質の人工的な発現を伴うながら、彼らは、特定していないということです。 PLAの1つの主な利点は、それがFRに匹敵する信号を生成するために必要な近さの程度と、組織内で互いに近接していると可能性が高い複合体を形成する内因性タンパク質の相互作用因子の細胞内局在を決定することができるということであるETとBIFC。 PLA起因抗体認識およびDNA増幅のカップリングに高い特異性と感度との相互作用を検出することができる。したがって、このアッセイは、相互作用の正確な位置を明らかに涙の形で個別の、明るい信号を生成することができる。また、ほとんどの可視抗原を検出することができる。最後に、PLAは、実行する比較的簡単な技術ではない、それが完了するために、標準的な免疫組織化学的手順よりも、もはやかかる。従って、PLAは、多くの場合、長い準備時間と広範なトラブルに悩まされている他のタンパク質 - タンパク質相互作用アッセイを介して技術的な利点を提供する。 このプロトコルは、PLAは、 その場での内因性のタンパク質-タンパク質相互作用を検出するために、ショウジョウバエ幼虫NMJに適用することができる方法を示す。ここでは、PLAは、は、Dlgおよびフーバーが実際のNMJのシナプス後領域に複合体中に存在することが示されている幼虫の体壁筋調製物に対して行われる。 PLAは、以前に幼虫のNMJを研究するために使用されていなかった、とショウジョウバエの組織でこのアッセイを使用している論文のほんの一握りは、現状であります。これは、 ショウジョウバエコミュニティにPLAのさらなる暴露は、他のより一般的に使用されるタンパク質-タンパク質相互作用アッセイを補完する追加のツールとしての利用増加につながることが期待されている。

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Name of Material/Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:191px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Forceps (fine #5)</td>
			<td style="width:120px;">Almedic</td>
			<td>A10-704</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:211px;">Sylgard Disc</td>
			<td style="width:120px;">World Precision Instruments</td>
			<td>SYLG184</td>
			<td style="width:191px;">Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Minutien Pins (0.0125 mm tip diameter)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td>26002-10</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Microdissection Scissors (ultra fine)</td>
			<td style="width:120px;">Fine Science Tools</td>
			<td style="width:111px;">15200-00</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">Platform Slides</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a &#60;20mm gap in between for sample mounting.</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">w1118</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">3605</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:211px;">hts01103</td>
			<td style="width:120px;">Bloomington Drosophila Stock Center</td>
			<td style="width:111px;">10989</td>
			<td style="width:191px;">Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Bouin&#39;s Solution</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">HT10132</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="114">
			<td height="114" style="height:114px;width:211px;">4% PFA (Paraformaldehyde): 4% PFA in 1x PBS</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Triton X-100 (Sigma-Aldrich - T8787)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">BSA (Bioshop Canada - ALB001). Store at 4 &#176;C.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Dlg (Discs large)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td style="width:191px;">Use at a 1:10 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">rabbit anti-HtsM (Hu-li tai shao)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">rabbit anti-Pak (p21-activated kinase)</td>
			<td style="width:120px;"></td>
			<td style="width:111px;"></td>
			<td style="width:191px;">Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">mouse anti-Wg (Wingless)</td>
			<td style="width:120px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:111px;">4D4</td>
			<td style="width:191px;">Use at a 1:5 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">goat anti-Hrp (Horseradish peroxidase)</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">123-065-021</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">FITC-conjugated donkey anti-goat</td>
			<td style="width:120px;">Jackson&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ImmunoResearch</td>
			<td style="width:111px;">705-095-003</td>
			<td style="width:191px;">Use at a 1:200 dilution in 1% BSA.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-mouse MINUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92004</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ PLA Probe anti-rabbit PLUS</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO92002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Detection Reagents Red</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td>DUO92008</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82049</td>
			<td style="width:191px;">Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:211px;">Duolink In Situ Mounting Medium with DAPI</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:111px;">DUO82040</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

detection of in situ protein-protein complexes at the drosophila larval neuromuscular junction using proximity ligation assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

野生型の第3齢幼虫のNMJでは、Dlgというは、主にタイプが終末であるよりは、Dlg免疫反応性レベルはIb型終末においてより顕著であることで、I型グルタミン酸作動性神経繊維末端のシナプス後膜で発見された（ 図3A）4。 HTSが筋肉全体で存在するが、両方のI型繊維末端に等しい登場フーバー免疫反応性レベルのシナプス後の領域に集中し、また、（ 図3A &#39;）...

Watch this Scientific Journal Video about Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay at JoVE.com

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

このプロトコルは、近接ライゲーションアッセイは、ショウジョウバエの幼虫神経筋接合部で、その場のタンパク質-タンパク質相互作用に検出するために使用することができる方法を示しています。この技術では、大規模で胡-LIの太極拳シャオディスクは、シナプス後の領域に、以前に共免疫沈降を介して同定関連を複合体を形成することが示されている。

の検出<em&gt;その場で</emで&gt;タンパク質 - タンパク質複合体<em&gt;ショウジョウバエ</em近接ライゲーションアッセイの使用&gt;幼虫神経筋接合部

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

detection-situ-protein-protein-complexes-at-drosophila-larval

Research

JoVE Journal

Neuroscience

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

13.0K ビュー.  Simon Fraser University. このプロトコルは、近接ライゲーションアッセイは、ショウジョウバエの幼虫神経筋接合部で、その場のタンパク質-タンパク質相互作用に検出するために使用することができる方法を示しています。この技術では、大規模で胡-LIの太極拳シャオディスクは、シナプス後の領域に、以前に共免疫沈降を介して同定関連を複合体を形成す?...

ビデオ: Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3の幼虫分節神経の可視化 RD齢ショウジョウバエ幼虫準備

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

神経科学

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

ショウジョウバエ幼虫分節軸索内ではシナプス小胞の in vivo可視化で

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

で、アクティブなゾーンの解剖とイメージングショウジョウバエ神経筋接合部

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

シーケンシャル神経筋接合部におけるシングルシュワン細胞の輪郭を描くのにフォトブリーチング

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

なぜ定量事項：での表現型のキャラクタリゼーション<em&gt;ショウジョウバエ</em&gt;幼虫の神経筋接合部

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

神経筋接合部機能を測定

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

高スループットとのマルチパラメトリック定量化のための2個のアルゴリズム<em&gt;ショウジョウバエ</em&gt;神経筋接合部形態

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

ショウジョウバエ幼虫神経筋接合部のシナプス電流の焦点 Macropatch 録音

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

認識された運動ニューロンの神経筋接合部における変化を検出する ショウジョウバエ 成脚の解剖と免疫染色

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

マウスバガル・アファレントニューロンにおけるGタンパク質結合受容体発現 の 検出

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...