Name: techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes
Uploaded: 2016-10-31T15:00:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes at JoVE.com

We thank Christian Lehner for providing the CENP-C antibody and Eric Joyce for recommendations on FISH. Work in the McKim lab was funded by a grant from NIH (GM101955).

注：特に断りのない限り、手順は室温で行われます。温度制御インキュベータは、ハエの飼育のための温度を維持するために使用され、特に断りのない限り、交差しています。 1.準備<ol><li>ハエを準備します。 <ol><li>前中期に富む卵母細胞のコレクション。 <ol><li>クリア大人は、健康な、若い株式またはクロス培養から飛びます。 25℃で2日間年齢ボトル。 メモ：詳細は、いくつかのクロス文化のために必要とされるかもしれないが、一般的に2人の健康のボトルは、十分であろう。 </li><li> 2日後、ボトルから（この時点では0〜2日齢である）〜100〜300人の女性を集めます。女性が処女である必要はありません。バイアルの側に酵母ペーストのDABを追加し、yeastedバイアルに30人の女性と10〜15人の男性それぞれに配置します。 25℃で2日間年齢バイアル。 </li></ol></li><li>中期に富む卵母細胞のコレクション。 <ol><li>株式やクロスculturから約100〜300人の女性を集めますエス。バイアルの側に酵母ペーストのDABを追加し、yeastedバイアルに30人の女性（無添加の男性と）それぞれを配置します。 25℃で3〜5日間年齢バイアル。 </li></ol></li></ol></li><li>ソリューションを準備します。 注：特に断りのない限り、溶液は、室温で無期限に保存してもよいです。 <ol><li> 500mMのHEPES、500mMのスクロース、275 mM酢酸ナトリウム、200mMの酢酸カリウム、50mMのグルコース、6mMの塩化マグネシウム、および5mM塩化カルシウム：修飾ロブの緩衝液（5×）を調製します。 7.4にpHを水酸化カリウム：8水酸化ナトリウム：10 N 11を使用してください。濾過により滅菌します。オートクレーブはありません。 -20℃で保管してください。 〜1×ロブの卵母細胞あたりの準備を200mlを調製するために、必要に応じて解凍します。 </li><li>固定ソリューション。 <ol><li>オプション＃1：ホルムアルデヒド/ヘプタンの固定を準備します。 1×リン酸緩衝生理食塩水（PBS）を加えた150mMのスクロース：固定バッファを準備します。使用するには、687.5μlの固定バッファおよび312.5μlの16％ホルムアルデヒドで新鮮作ります卵母細胞の準備あたり。 注意：ドラフト内でホルムアルデヒド溶液を使用しながら、手袋を着用してください。機関のガイドラインに従って廃棄処分してください。 </li><li>オプション＃2：ホルムアルデヒド/カコジル酸固定を準備します。修正ミックスを準備：250mMのスクロース、100mMの酢酸カリウム（pHは7.5）、25 mM酢酸ナトリウム（pH 7.0）、および25mM EGTA（pHは8.0）。使用するには、400μlの新鮮なミックスを修正行い、100μlのカリウムカコジル（1 M、pHは7.2）、および卵母細胞の準備あたり500μlの16％ホルムアルデヒド。 注意：カリウムカコジルはヒ素が含まれています。 </li></ol></li><li> 1×PBS + 1％または0.05％のトリトンX-100：PBS /トリトンX-100を準備します。 4℃で保存。 </li><li> 1×PBS、（w / v）の0.5％のBSA、および0.1％のTween-20：PBS-Tween 20で、ウシ血清アルブミン（BSA）（PTB）を準備します。一週間4℃で保存してもよいです。 </li><li> in situハイブリダイゼーション（FISH）ソリューションでは 、蛍光。 <ol><li>調製20X塩化ナトリウム、クエン酸ナトリウム（SSC）：3 M塩化ナトリウムおよび0.3 Mクエン酸ナトリウムを。 </li><li>調製2×SSC、トゥイーン20（SSCT）：2×SSC + 0.1％のTween-20。卵母細胞の準備あたり新鮮な、〜20ミリリットルを加えます。 </li><li>準備ホルムアミド溶液：2×SSC、0.1％Tween-20を加えホルムアミド。 1 mlの20％ホルムアミド0.5mlの40％ホルムアミド、および卵母細胞の準備につき2 50mlの％ホルムアミド、新鮮します。 注意：ドラフト内でホルムアミド溶液を使用しながら、手袋を着用してください。機関のガイドラインに従って廃棄処分してください。 </li><li> （w / v）の2×SSC、50％ホルムアミド、10％硫酸デキストラン：ハイブリダイゼーション溶液を調製します。 4℃で保存。 </li></ol></li><li> FISHプローブ。 <ol><li>注文HPLC精製を有するオリゴヌクレオチド（配列については表1を参照）、5 &#39;蛍光修飾（ 例えば 、Cy3またはCy5の）を希望します。 50 ngの/μlのでトリスEDTA（TE）で再懸濁します。 注：すべての回で、露光からオリゴを保護します。 </li></ol></li></ol></li></ol>後期ショウジョウバエ卵母細胞の2コレクション<ol><li>として<eメートル&gt;プラスチックやガラスに固執する、無傷の膜を有するショウジョウバエの卵母細胞、プレコート1 5ミリリットルチューブとPTBと卵母細胞の準備につき1パスツールピペットの内部。 </li><li>二酸化炭素がすべて〜100〜300 yeastedハエを麻酔し、〜100ミリリットル1×ロブのバッファーを含むブレンダーに追加します。パルス3回（約1秒ずつ）。活性化を避けるために、&lt;20分間ロブの中で卵母細胞を保管してください。 注：別の方法として、卵母細胞が雌から手で解剖することができます。この方法の利点は、以下の女性を必要とすることです。しかし、ケアは、低酸素状態7に関連したアーチファクトを回避するためにわずか数分に二酸化炭素への曝露を制限するように注意する必要があります。 </li><li>大きな身体の部分を除去するために、250ミリリットルのビーカーに大きなメッシュ（〜1500ミクロン）を通して濾過。多くの無傷の腹部には、追加のロブのバッファを使用して、メッシュ、再研削材上に残り、再びフィルタする場合。その後、できるだけ大きな体の部分の多くを取り除く、最上層を吸引する、〜2分の決済をしてみましょう。 </li><li> Fiの250ミリリットルのビーカーに小さなメッシュ（〜300ミクロン）を通してLTER。追加のロブのコーティングされたパスツールピペットを用いて、第1のビーカーのうち残りの卵母細胞を洗浄します。整が〜3分してみましょう。卵母細胞が沈降します。すべてしかし〜10ミリリットルを吸引除去。 </li><li>コーティングされた5ミリリットルのチューブに収まるように10ミリリットルの限りを注ぎます。 、落ち着く液体を除去し、残りは繰り返してみましょう。追加のロブのコーティングされたパスツールピペットを用いてビーカーのうち、残りの卵母細胞を洗浄します。 〜3-5分間5ミリリットルチューブに定住してみましょう。 </li></ol> 3.薬品による治療（オプション） <ol><li>コート各卵母細胞の準備のためのPTBと第2の5 mlのチューブ。各卵母細胞分取（ 表2）のために1ミリリットルロブのそれぞれに適切な溶媒（対照）または薬剤を追加します。 </li><li>第二のコーティングされた5ミリリットルチューブに分割した卵母細胞。沈降は、液体を除去し、1チューブ及び第二のチューブに1ミリリットルロブのプラス薬物に1ミリリットルロブのプラス溶剤を追加してみましょう。薬物治療（ 表2）のための適切な時間のために章動させます。 Lらが落ち着きます。 </li></ol> 4.固定<ol><li>吸引し、オフすべての液体とすぐに1ミリリットル修正を加えます。 </li><li>オプション＃1：ホルムアルデヒド/ヘプタン固定（固定緩衝液+ 5％ホルムアルデヒド）。 <ol><li>ニューテーター上で2.5分間固定してください。 1ミリリットルのヘプタン及びボルテックス1分を追加します。 〜1分で沈降してみましょう。 </li><li>すべての液体を除去し、その後PBS 1X 1ミリリットルを追加します。ボルテックス30秒。 〜1分で沈降してみましょう。 </li><li>すべての液体を除去し、次いで1×PBSでチューブを埋めます。 注：卵母細胞は、直ちに使用するか、または室温で数時間ニューテーター上で維持することができます。 </li></ol></li><li>オプション＃2：ホルムアルデヒド/カコジル固定（ミックスプラス8％のホルムアルデヒド及び100mMのカコジルを修正）。 <ol><li>ニューテーター上で6分間固定してください。 2分の決済をしてみましょう。液体を除去し、次いで1×PBSでチューブを埋めます。 注：卵母細胞は、直ちに使用するか、または室温で数時間ニューテーター上で維持することができます。 </li></ol></li></ol> 5.取り外し膜（「ローリング」） <ol><li>コーティングされたパスツールピペットを用いて、スライドガラスのつや消し（サンドブラスト）部分に〜500〜1,000卵母細胞を追加します。鉗子を使用して、すべてのボディパーツや異物を取り除きます。卵母細胞を乾燥させないでください。必要に応じて1×PBSを追加します。 </li><li>すべての膜が除去されるまで（卵母細胞全体にカバースリップの端をドラッグすると、最も効果的に機能します。）卵母細胞およびそっと「ロール」卵母細胞の上にカバースリップを置き、定期的に必要に応じてより多くの1×PBSを追加し、顕微鏡下で進行状況を確認します。あまりにも多くの圧力が卵母細胞を破壊するように注意してください。 注：唯一の免疫蛍光のために、（または免疫蛍光なし）FISHについては、ステップ6に進み、ステップ7に進みます。 </li></ol> ショウジョウバエ卵母細胞の6抗体染色<ol><li>抽出とブロッキング<ol><li> 〜15ミリリットルのPBS / 1％トリトンX-100を含む15ミリリットルコニカルチューブに巻かれた卵母細胞を洗浄します。無未満1.5時間この溶液を超えない2時間で章動卵母細胞。このステップでは、抗体の浸透を可能にします。 </li><li>卵母細胞は、〜2分に解決しましょう。 PBS / 0.05％トリトンX-100を追加し、できるだけ多くの膜と一緒に液体を削除します。 注：膜は、圧延卵母細胞よりも遅いセトリングします。 </li><li>卵母細胞は、〜2分に解決しましょう、その後、液体のすべてが、〜1ミリリットルを削除します。転送卵母細胞を1.5 mlチューブを卒業し、残りの液体を除去します。 1時間ブロッキングし、うなだれるのために1ミリリットルPTBを追加します。 </li></ol></li><li>抗体染色<ol><li>胚に対する二次抗体の予備吸収。 注：この手順は、ショウジョウバエのタンパク質との非特異的抗体相互作用からバックグラウンド染色を排除します。 <ol><li>典型的な手順8あたりのショウジョウバエの胚（〜25μl）を収集し、固定します。 -20℃のメタノール中で保管してください。 </li><li>胚からメタノールを除去、800μlのメタノールを加えた200μlの1×PBSを追加し、15分間うなだれます。上清の500μLを削除し、500μlの1×PBSと交換。その後、15分間章動させます。 （洗浄の〜1時間の合計）を2回繰り返し、その後、PTBでフィニッシュ。 注：胚はすぐに使用するか、または室温で数時間ニューテーター上で維持することができます。 </li><li> （最終容量は300μlのであろうことを念頭に置いて、ステップ7.4を参照してください。）、液体を除去卵母細胞プレップあたり200μlにPTBで記入し、適切な希釈で蛍光標識された二次抗体を追加章動4℃で一晩（望ましいです）または3〜4時間、室温で。 注：前吸収抗体は、ステップ6.2.4で使用されます。 注：蛍光抗体が追加された後、できるだけ多くの暗闇の中でサンプルを保管してください。 </li></ol></li><li> 、卵母細胞から液体を除去する300μlにPTBで記入し、適切な希釈で一次抗体を追加します。 4℃での章動一晩（望ましい）または3〜4時間、室温で。 </li><li>洗浄は、1ミリリットルPTBで15分間、4回ずつ卵母細胞。 </li><li>削除します卵母細胞からの液体、胚（プレ吸収二次抗体）からの上清200μlを添加します。その後、300μlにPTBでいっぱい。 3〜4時間（好ましく）または4℃で一晩、室温で章動させます。 </li><li> 1ミリリットルPTBで15分間、一度卵母細胞を洗浄し、液体を除去。その後、7分間0.5μlのヘキスト33342と500μlのPTBとうなだれるを追加します。 </li><li> 1ミリリットルPTBで15分間ずつ二回卵母細胞を洗ってください。 注：卵母細胞は直ちにスライド上に取り付けられてもよいし、撮像のための準備ができるまで4℃でPTBに格納されてもよいです。 </li></ol></li></ol> 7. FISH（上記の手順5から続けます） <ol><li> 〜15ミリリットル2倍SSCTを含む15ミリリットルコニカルチューブに巻かれた卵母細胞を洗浄します。卵母細胞は、〜2分に解決しましょう。できるだけ多くの膜と一緒に液体すべてが、〜0.5ミリリットルを削除します。 注：膜は、圧延卵母細胞よりも遅いセトリングします。 </li><li>転送は、0.5 mlチューブに卵母細胞および残りの液体を除去します。続いて500μlのを追加および削除20％、40％、および50％ホルムアミドの溶液を、各溶液中に10分間章動。 注：卵母細胞は、ホルムアミドの割合が高いと遅く沈降します。 </li><li> 500μlの50％ホルムアミド溶液を加えた後、液体の削除、および1〜5時間、37℃でうなだれます。 注：インキュベーション時間が長いほど、より良いプローブの浸透につながります。 </li><li>これ以上の100〜よりμlの卵母細胞を残して、液体を除去し、36μlのハイブリダイゼーション溶液を加えたプローブの2μlの（50 ngの/μL）と水の2μlあるいは2 番目のプローブ（ 表1）の2μlを添加します。 </li><li>一晩37℃の水浴中でインキュベートし、続いて3分間（FISHのみ）または20分間（FISHプラス免疫蛍光法）のために80°C、91°Cでインキュベートします。 注：これらの手順は、サーモサイクラーで行うことができます。 </li><li>液体を除去しないでください。 500μlの1時間、37℃で50％ホルムアミド溶液と、うなだれるを追加します。 </li><li> 、液体を除去、沈降をしてみましょう500μlの50％ホルムアミド溶液を加え、1時間37℃でdはうなだれます。 </li><li>落ち着くしましょう、液体を除去、500μlの20％ホルムアミド溶液を添加し、10分間室温でうなだれます。 </li><li> 500μlの2×SSCT（沈降が、その後、液体を追加し、数回転倒、繰り返しを削除しましょう。）が沈降してみましょう、液体を除去し、4時間500μlのPTBとうなだれるを追加で3クイック回の洗浄を行います。 </li></ol> 8.抗体染色FISH後<ol><li> 、卵母細胞から液体を除去する300μlにPTBで記入し、FITCにコンジュゲート10μlの抗αチューブリン抗体を追加します。あるいは、他の抗体は、上記のプロトコルに従って、使用することができます。室温で一晩章動させます。 </li><li>その後、7分間0.5μlのヘキスト33342と500μlのPTBとうなだれるを追加し、液体を除去し、500μlのPTBとの15分間に一度卵母細胞を洗浄します。 </li><li> 500μlのPTBで15分間ずつ二回卵母細胞を洗ってください。 注：卵母細胞は直ちにスライド上に取り付けられてもよいし、準備が整うまで4℃でPTBに格納することができますイメージング。暗闇の中で卵母細胞を蛍光抗体が追加された後、可能な限りにしてください。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="1in"> 繰り返し名前 </td><td fo:width="1in"> 染色体 </td><td> オリゴ配列* </td></tr><tr><td> 359 </td><td>バツ</td><td> GGGATCGTTAGCACTGGTAATTAGCTGC </td></tr><tr><td> AACAC </td><td> 2 </td><td> AACACAACACAACACAACACAACACAACACAACACAACAC </td></tr><tr><td>ドデカ</td><td> 3 </td><td> CCCGTACTGGTCCCGTACTCGGTCCCGTACTCGGT </td></tr><tr><td> 1.686 </td><td> 2 + 3 </td><td> AATAACATAGAATAACATAGAATAACATAG </td></tr><tr><td> AATAT </td><td> 4（+ Y） </td><td> AATATAATATAATATAATATAATATAATAT </td></tr><tr><td colspan="3"> *エリック・ジョイス、パーソナル通信、サリバンらから他の配列から359の配列。8 </td&gt; </tr></tbody></table> 表1： ショウジョウバエ 動原体繰り返し のためのFISHプローブ 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ドラッグ </td><td> 溶媒 </td><td> ストック濃度 </td><td> 最終濃度 </td><td> 治療の時間 </td><td> 効果 </td></tr><tr><td>コルヒチン</td><td>エタノール</td><td> 125 mMの</td><td> 150μM </td><td> 10分または30分</td><td>非動原体（10分）9またはすべて（30分）微小管を不安定にします</td></tr><tr><td>パクリタキセル</td><td> DMSO </td><td> 10 mMの</td><td> 10μM </td><td> 10分</td><td>マイクを安定させますrotubules </td></tr><tr><td> Binucleine 2 </td><td> DMSO </td><td> 25 mMの</td><td> 25μM </td><td> 20分</td><td>オーロラBキナーゼ10を阻害</td></tr></tbody></table> 表2：薬物治療。

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+a+role+for+heterochromatin+in+meiotic+chromosome+segregation.">Direct evidence of a role for heterochromatin in meiotic chromosome segregation.</a> Cell. 86 (1), 135-146 (1996).</li><li>Gilliland, W. D., Hughes, S. F., Vietti, D. R., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congression+of+achiasmate+chromosomes+to+the+metaphase+plate+in+Drosophila+melanogaster+oocytes.">Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes.</a> Dev Biol. 325 (1), 122-128 (2009).</li><li>Gilliland, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+transiently+sequesters+mps1+and+polo+to+collagenase-sensitive+filaments+in+Drosophila+prometaphase+oocytes.">Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes.</a> PLoS One. 4 (10), e7544 (2009).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Radford, S. J., Hoang, T. L., G&#322;uszek, A. A., Ohkura, H., McKim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+and+End-On+Kinetochore+Attachments+Are+Coordinated+to+Achieve+Bi-orientation+in+Drosophila+Oocytes.">Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.</a> PLoS Genet. 11 (10), e1005605 (2015).</li><li>Smurnyy, Y., Toms, A. V., Hickson, G. R., Eck, M. J., Eggert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binucleine+2,+an+isoform-specific+inhibitor+of+Drosophila+Aurora+B+kinase,+provides+insights+into+the+mechanism+of+cytokinesis.">Binucleine 2, an isoform-specific inhibitor of Drosophila Aurora B kinase, provides insights into the mechanism of cytokinesis.</a> ACS Chem Biol. 5 (11), 1015-1020 (2010).</li><li>Mahowald, A. P., Goralski, T. J., Caulton, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activation+of+Drosophila+eggs.">In vitro activation of Drosophila eggs.</a> Dev Biol. 98 (2), 437-445 (1983).</li><li>Page, A. W., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+meiotic+divisions+in+Drosophila+oocytes.">Activation of the meiotic divisions in Drosophila oocytes.</a> Dev Biol. 183 (2), 195-207 (1997).</li><li>Tavosanis, G., Llamazares, S., Goulielmos, G., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+gamma-tubulin+in+the+acentriolar+female+meiotic+spindle+of+Drosophila.">Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.</a> EMBO J. 16 (8), 1809-1819 (1997).</li><li>Endow, S. A., Komma, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+dynamics+during+meiosis+in+Drosophila+oocytes.">Spindle dynamics during meiosis in Drosophila oocytes.</a> J Cell Biol. 137 (6), 1321-1336 (1997).</li><li>Matthies, H. J., Clarkson, M., Saint, R. B., Namba, R., Hawley, R. S., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , 67-85 (2000).</li><li>Colombi&#233;, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+roles+of+Incenp+crucial+to+the+assembly+of+the+acentrosomal+metaphase+spindle+in+female+meiosis.">Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis.</a> Development. 135 (19), 3239-3246 (2008).</li></ol>

The authors have nothing to disclose.

ショウジョウバエの卵母細胞ステージング 細長いカリオソームは、多くの場合、中期卵母細胞から前中期区別するためにカリオソーム形状を使用して、前中期卵母細胞に見られるが問題となる可能性があります。前中期の間、カリオソームは、丸い形状として始まり伸長し、その後卵母細胞が中期停止に近づくにつれて円形に後退します。これは、?...

The study of meiosis is sometimes described as the "genetics of genetics". This is because the fundamental properties of chromosome inheritance and independent assortment are carried out through the segregation of chromosomes during gamete production. An important demonstration of the chromosome theory of inheritance came in 1916 from the work of Calvin Bridges in Drosophila melanogaster1. This and other classical genetics studies in Drosophila contributed greatly to our understanding of genetics. Cytological examination of meiotic chromosomes in Drosophila oocytes, however, has been challenging. This is primarily because immunofluorescence of late-stage Drosophila oocytes, when the spindle assembles and chromosomes are oriented for segregation, is hampered by the presence of membranes that render the oocyte impenetrable to antibodies.
Despite this challenge, Drosophila oocytes remain an attractive model for the study of chromosome behavior and spindle assembly. This is because of the powerful genetic tools available in Drosophila, but also because the oocytes arrest at metaphase I, when the chromosomes are oriented and the spindle is fully formed. This facilitates the collection and examination of large numbers of oocytes at this important stage of cell division. In addition, a simple model organism that is amenable to genetic manipulation for the study of oocyte chromosome segregation can provide an important contribution to our understanding of human reproductive health. Errors in chromosome number are the leading genetic cause of miscarriage and birth defects in humans2. A majority of these errors can be traced to the oocyte and are correlated with increasing maternal age. The average age of mothers in the U.S. has been increasing, making this a major public health concern.
We describe here methods for the cytological examination of Drosophila oocytes, including a demonstration of how to remove the oocyte membranes. These methods are modifications of protocols first described by Theurkauf and Hawley3, Zou et al.4, and Dernburg et al.5. We also include methods for the enrichment of different stages of oocytes, based on a protocol first described by Gilliland et al.6. Finally, we add instructions for the drug treatment of Drosophila oocytes. Together, these methods allow the cytological investigation of oocyte chromosome segregation and spindle assembly in Drosophila.

<table><tbody><tr><td>15 ml conical tubes</td><td>Various</td><td></td><td></td></tr><tr><td>16% formaldehyde</td><td>Ted Pella, Inc.</td><td>18505</td><td>HAZARDOUS; once opened, discard after one month</td></tr><tr><td>250 ml beakers</td><td>Various</td><td></td><td></td></tr><tr><td>5 ml tubes</td><td>Various</td><td></td><td></td></tr><tr><td>active dry yeast</td><td>Various</td><td></td><td>mix with water to make a paste the consistency of peanut butter</td></tr><tr><td>anti-&amp;alpha;-tubulin antibody conjugated to FITC</td><td>Sigma</td><td>F2168</td><td>clone DM1A</td></tr><tr><td>Binucleine 2</td><td>Sigma</td><td>B1186</td><td>HAZARDOUS</td></tr><tr><td>blender</td><td>Various</td><td></td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A4161</td><td></td></tr><tr><td>calcium chloride</td><td>Various</td><td></td><td></td></tr><tr><td>colchicine</td><td>Sigma</td><td>C-9754</td><td>HAZARDOUS</td></tr><tr><td>coverslips</td><td>VWR</td><td>48366-227</td><td>No. 1 1/2</td></tr><tr><td>dextran sulfate</td><td>Various</td><td></td><td></td></tr><tr><td>DMSO</td><td>Various</td><td></td><td></td></tr><tr><td>EGTA</td><td>Various</td><td></td><td></td></tr><tr><td>ethanol</td><td>Various</td><td></td><td></td></tr><tr><td>forceps</td><td>Ted Pella, Inc.</td><td>5622</td><td>Dumont tweezers high precision grade style 5</td></tr><tr><td>formamide</td><td>Sigma</td><td>47670-250ML-F</td><td></td></tr><tr><td>glass slides</td><td>VWR</td><td>48312-003</td><td></td></tr><tr><td>glucose</td><td>Various</td><td></td><td></td></tr><tr><td>graduated 1.5 ml tubes</td><td>Various</td><td></td><td></td></tr><tr><td>HEPES</td><td>VWR</td><td>EM-5330</td><td>available from several venders</td></tr><tr><td>Hoechst 33342</td><td>Various</td><td></td><td></td></tr><tr><td>magnesium chloride</td><td>Various</td><td></td><td></td></tr><tr><td>methanol</td><td>Various</td><td></td><td></td></tr><tr><td>large mesh (~1,500 &amp;micro;m)</td><td>VWR</td><td>AA43657-NK</td><td>variety of formats and other suppliers, 12 or 14 mesh</td></tr><tr><td>small mesh (~300 &amp;micro;m)</td><td>Spectrum labs</td><td>146 424</td><td>variety of formats, &lt;em&gt;e.g.&lt;/em&gt;, 146 422 or 146 486</td></tr><tr><td>nutator</td><td>Various</td><td></td><td></td></tr><tr><td>Pasteur pipets</td><td>Various</td><td></td><td></td></tr><tr><td>potassium acetate</td><td>Various</td><td></td><td></td></tr><tr><td>Cacodylic acid</td><td>Sigma</td><td>C0125</td><td>HAZARDOUS; alternatively, sodium cacodylate may be substituted</td></tr><tr><td>potassium hydroxide</td><td>Various</td><td></td><td></td></tr><tr><td>sodium acetate</td><td>Various</td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Various</td><td></td><td></td></tr><tr><td>sodium citrate</td><td>Various</td><td></td><td></td></tr><tr><td>sodium hydroxide</td><td>Various</td><td></td><td></td></tr><tr><td>sucrose</td><td>Various</td><td></td><td></td></tr><tr><td>taxol (paclitaxel)</td><td>Sigma</td><td>T1912</td><td>HAZARDOUS</td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>PI-28314</td><td></td></tr><tr><td>Tween 20</td><td>Fisher</td><td>PI-28320</td><td></td></tr><tr><td>vortex</td><td>Various</td><td></td><td></td></tr></tbody></table>

techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

我々がここで説明している方法は、減数分裂の三段階を表す後期ショウジョウバエ卵母細胞の収集（ 図1）になります。前期中の卵母細胞はカリオソーム（ 図1A）を取り囲む領域におけるチューブリンシグナルの欠如によって可視である、核膜の存在によって区別されます。前中期は、核膜の崩壊時のスピンドル組み立て後の期間です?...

Watch this Scientific Journal Video about Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes at JoVE.com

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

固定における減数分裂Iのイメージング前中期および中期のための技術<em&gt;ショウジョウバエ</em&gt;卵母細胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

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Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Unit 2

Meiosis

SCIENTISTS IN ACTION

9.0K ビュー.  Rutgers University. We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

ビデオ: Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

マウス卵母細胞を減数分裂の間に人間の遺伝子の機能を評価するために使用私

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

遺伝学

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Prometaphase の腕凝集度と中期の状態を監視する蛍光In Situハイブリダイゼーション (魚) を使用して私はショウジョウバエの卵母細胞

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

発生生物学

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

生物学

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

ショ ウジョウバエ・ ウーサイトにおける核移動

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment and maintenance of epithelial tissue morphology and organ morphogenesis. Moreover, the BM is essential for tissue modeling, serving as a signaling platform, and providing external forces to shape tissues and organs. Despite the many important roles that the BM plays during normal development and pathological conditions, the biological pathways controlling the intracellular trafficking of BM-containing vesicles and how basal secretion leads to the polarized deposition of BM proteins are poorly understood. The follicular epithelium of the Drosophila ovary is an excellent model system to study the basal deposition of BM membrane proteins, as it produces and secretes all major components of the BM. Confocal and super-resolution imaging combined with image processing in fixed tissues allows for the identification and characterization of cellular factors specifically involved in the intracellular trafficking and deposition of BM proteins. This article presents a detailed protocol for staining and imaging BM-containing vesicles and deposited BM using endogenously tagged proteins in the follicular epithelium of the Drosophila ovary. This protocol can be applied to address both qualitative and quantitative questions and it was developed to accommodate high-throughput screening, allowing for the rapid and efficient identification of factors involved in the polarized intracellular trafficking and secretion of vesicles during epithelial tissue development.

Confocal and Super-Resolution Imaging of Polarized Intracellular Trafficking and Secretion of Basement Membrane Proteins During Drosophila Oogenesis

The basement membrane is essential for tissue and organ morphogenesis during development. To better understand the mechanisms leading to proper placement of this structure, the protocol presented describes methods to visualize and characterize the intracellular trafficking and secretion of basement membrane proteins in epithelial cells using confocal and super-resolution microscopy.

ショウジョウバエの卵子形成期における分極細胞内輸送と基底膜タンパク質の分泌の共焦点および超解像イメージング

The basement membrane (BM) - a specialized sheet of extracellular matrix present at the basal side of epithelial cells - is critical for the establishment ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

リビングでの動的動原体タンパク質の挙動と染色体の動きを図解して有糸分裂チェックポイントを勉強<em&gt;ショウジョウバエ</em&gt;合胞体胚

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

でGFP標識タンパク質のイメージングを生きる<em&gt;ショウジョウバエ</em&gt;卵母細胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子形成細胞学的分析：ライブとの固定の準備<em&gt;ショウジョウバエ</em&gt;精巣

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Preparation of Fixed Drosophila Oocytes for Immunostaining: A High-Throughput Method to Fix and Remove the Outer Membrane

Source: Radford, S. J., McKim, K. S. <a href="https://jove.unicartagenaproxy.elogim.com/video/54666/" target="_blank">Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes</a>. J. Vis. Exp. (2016).
Immunostaining of Drosophila late-stage oocytes can prove challenging due to the oocytes&#39; surrounding membranes. This video describes a high-throughput method for obtaining oocytes, their fixation, and membranes&#39; removal for immunostaining. The featured protocol demonstrates these techniques with late-stage oocytes used to visualize different stages of meiosis.

免疫染色のための固定ショウジョウバエ卵子の調製:外膜を固定および除去するためのハイスループット法

Source: Radford, S. J., McKim, K. S. Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes. J. Vis. Exp. (2016).
...

固定における減数分裂Iのイメージング前中期および中期のための技術

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この動画の章

マウス卵母細胞を減数分裂の間に人間の遺伝子の機能を評価するために使用私

Prometaphase の腕凝集度と中期の状態を監視する蛍光In Situハイブリダイゼーション (魚) を使用して私はショウジョウバエの卵母細胞

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

ショウジョウバエ・ウーサイトにおける核移動

ショウジョウバエの卵子形成期における分極細胞内輸送と基底膜タンパク質の分泌の共焦点および超解像イメージング

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

リビングでの動的動原体タンパク質の挙動と染色体の動きを図解して有糸分裂チェックポイントを勉強

でGFP標識タンパク質のイメージングを生きる

精子形成細胞学的分析：ライブとの固定の準備

免疫染色のための固定ショウジョウバエ卵子の調製:外膜を固定および除去するためのハイスループット法

固定における減数分裂Iのイメージング前中期および中期のための技術

要約

さらに動画を探す

この動画の章

マウス卵母細胞を減数分裂の間に人間の遺伝子の機能を評価するために使用私

Prometaphase の腕凝集度と中期の状態を監視する蛍光In Situハイブリダイゼーション (魚) を使用して私はショウジョウバエの卵母細胞

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

ショ ウジョウバエ・ ウーサイトにおける核移動

ショウジョウバエの卵子形成期における分極細胞内輸送と基底膜タンパク質の分泌の共焦点および超解像イメージング

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

リビングでの動的動原体タンパク質の挙動と染色体の動きを図解して有糸分裂チェックポイントを勉強

でGFP標識タンパク質のイメージングを生きる

精子形成細胞学的分析：ライブとの固定の準備

免疫染色のための固定ショウジョウバエ卵子の調製:外膜を固定および除去するためのハイスループット法

ショウジョウバエ・ウーサイトにおける核移動