IMは、マリーキュリーの再統合の助成金によってサポートされています。この作品は、イスラエルの科学基金グラントのないによってサポートされていました。 1146年から1108年、ヘレンと幹細胞科学のワイツマン研究所での研究とLeir慈善基金のためのマーティンキメル研究所による。

1。産卵<ol><li>解剖前の5日間：交尾雌は酵母を添加した新鮮な食品に2〜4時間の卵を産むことができる。同期化されるにとよく発達した幼虫、それは（約30卵/ 25ミリメートルバイアル）培養を混雑していることが重要です。通常は、7月16日女性は彼らが横たわってどれだけに応じて、バイアルごとに使用されています。 </li></ol> 2。幼虫の選択<ol><li>リンゲル培地（128mMのNaCl、2mMのKCL、1.8mmのCaCl 2で 、4mmのMgCl 2を 、35.5mMスクロース、5mMのHepes緩衝液pH 6.9）で満たされた9もガラス解剖皿を準備します。 </li><li>リンガーの培地を含む6ウェルプレートに細胞ストレーナーを準備し、氷の上に置きます。また、我々はナイロンメッシュを装着した特別に作られた金型を使用してください。 </li><li>微細な生物学的なピンセット（鉗子）を使用してバイアル壁から時限幼虫を選択し、リンゲルを含む解剖皿に置いてください。 </li><li>きれいよくする女性の幼虫を移す。我々は、彼らの生殖腺で、男性から女性を区別する。雄の精巣は、簡単に脂肪体の後部3分の1に埋め込まれた大きな明確な楕円形のように識別されます。脂肪体の同じ部分に位置する女性の卵巣は、、はるかに小さい、透明な、丸い球体として識別することができます。 </li></ol> 3。幼虫の解剖<ol><li>鉗子で脳へのちょうど後方幼虫を押しながら、鉗子の2番目のペアと頭を取り除く。 </li><li>気管が下向きで、背側の残りの後部を置きます。 </li><li>鉗子の一組と後部気門で幼虫を持ち、約半分幼虫脂肪体が現れるまで、他のペアが後方キューティクルをスライドさせている間に徐々に内側に押し込みます。 </li><li>しっかりと後端を保持し、緩くキューティクルを保持するために鉗子の他のペアを使用してください。静かにゆっくりと後端のようキューティクルとギャップを介して接続されている腸のスライドを引き出します。このプロセスの最後に、脂肪体は完全に腸とキューティクルから分離する必要があります。脂肪体の前部から腸を外します。染色しやすく取り付けを行うには、それは脂肪体がそのまま残ることが重要です。 </li><li>望ましいも含む解剖幼虫から、リンゲル培地で十分に牧草地のピペットを濡らす。これはコートピペットを助け、それにこだわってから脂肪体を防ぎます。セルストレーナーで氷冷リンゲル媒体に脂肪体を転送するために牧草地のピペットを使用してください。 </li></ol> 4。固定と染色すべてのステップは4℃、一次抗体とのインキュベーションを除いて、室温で行われる℃に<ol><li>リンゲル培地で5％ホルムアルデヒドで脂肪体をインキュベートする。穏やかに振盪しながら20分間。 </li><li> 1パーセントPBT（1％トリトンX - 100 PBS中）で5分間洗浄する。 10分や45分3回目ごとにこの手順を繰り返します。 1％トリトンX - 100は、幼虫の卵巣を穿孔し、抗体がそれに浸透できるようにするために必要です。 </li><li>穏やかに攪拌しながら1時間0.3％PBTB（0.3％トリトンX - 100およびPBS中の1％BSA）でブロック</li><li> 4℃で一晩0.3％PBTBで希釈して所望の1 回目の抗体°穏やかに振とうさせながらCでインキュベートする。このステップは通常、ローラーに0.2 mlチューブに実行されます。 </li><li>セルストレーナーに戻って体脂肪を転送します。 </li><li>穏やかに撹拌しながら0.3％PBTBで3回、30分ごとを、洗う。 </li><li>穏やかに攪拌しながら1時間5％正常ロバ血清を添加した0.3％PBTBでブロックする。 </li><li>穏やかに撹拌しながら2時間ブロッキング溶液（0.3％PBTで5％正常ロバ血清を補充した0.3％PBTB）に（製造業者の仕様に従って）希釈した適切な二次抗体でインキュベートする。抗体が蛍光である場合、これ以降から暗所でインキュベートする。このステップは通常、ローラーに0.2 mlチューブに実行されます。 </li><li> PBTは、穏やかに撹拌しながら0.3％、30分ごとに3回洗浄する。 </li></ol> 5。取り付け<ol><li>清潔なエッペンドルフチュー​​ブに脂肪体を転送する。非常に慎重に離れてすべての液体を除去し、直ちにVectashieldマウンティングメディアでカバー。卵巣は脂肪体から分離されている間、それが硬化しないので、我々は日常的にvectashieldを使用してください。 までの週のためのサンプルは4℃で固定メディアに格納することができます。 </li><li>ピペットチップの先端をカットし、慎重に顕微鏡スライド上にマウントするメディアの最小量（30ミリメートルのカバースリップのための約30μL）で脂肪体を転送するために使用します。 </li><li>脂肪体を分散さ0.1mmの直径のピンを保持する、二つのニッケルメッキピンホルダーを使用してください。卵巣は、脂肪体の後部3分の1程度で位置しています。それらを囲む脂肪体は通常、&quot;花&quot;のような形をしている。これは、卵巣の位置を特定するのに役立ちます。脂肪体の残りの部分から&quot;花&quot;をばらばらにすると、後者を捨てる。 </li><li>脂肪体のsurroを削除します。その周りに慎重に2つのピンを交配することにより生殖腺をunding。離れて脂肪体の残渣からのスライド上で分離された生殖腺を置きます。 </li><li>マニキュアでカバーガラスおよびシールでカバー。 </li><li>共焦点顕微鏡を用いて直接視覚化する。サンプルは、最大3週間まで4℃で保存することができる。 </li></ol> 6。代表的な結果我々はGSCsとその体細胞のニッチを含む、体の卵巣内にいくつかの細胞系譜の確立に従うように、上記のプロトコルを使用している。この目的のために我々は発展途上生殖腺における異なる細胞型を区別する特異的な抗体とマーカーを使用してください。ここでは、抗体のさまざまな組み合わせで染色されたtwo LL3卵巣の例を示します。図1Aは、端末のフィラメントと一緒にニッチの体細胞を形成するキャップ細胞（緑）、強調表示されます。図1Bは、直接生殖細胞（青）に連絡混在細胞を（ICは、マゼンタ）、を示しています。 <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="図1" /> 図1。LL3卵巣。 （A）モノクローナル抗体1B1（マゼンタ）は体細胞と汚れfusomeを、始原生殖細胞内で細胞内小器官（矢印）を概説します。エンハンサートラップHH - lacZを（抗β-ガラクトシダーゼ、緑色）は、端末のフィラメントで表されます。フィラメント体キャップ細胞（矢頭）の基部に観察することができます。 （B）1B1抗体（緑）は卵巣内のすべての体細胞を概説。抗ヴァーサ（青）は、すべての始原生殖細胞にラベルを付けます。 PGCは直接混在細胞を（ICは、抗交通渋滞、マゼンタ）にお問い合わせください。バーは、（AおよびB用）20μmである。

<ol>
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利害の衝突は宣言されません。

このビデオでは、後期三齢幼虫卵巣の単離と染色プロトコルを示しています。日常的にかつ確実にこのプロトコルを実行するには、注意が以下の点に注意を払う必要があります。 <ol><li>同期とよく発達した幼虫の場合は、上の混雑を避ける必要があります。 </li><li>小さな半透明の卵巣の損失を避けるために、脂肪体はそのまま分析することを確認してください。また、これは（第2または?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kcl</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Albumin Bovine Fraction V</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>160069</td>
<td>&nbsp;</td>
<tr>
<td>Dumont biology tweezers 5 dumstar polished</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Nickel plated pin holder</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>s.s minutien pins 0.1mm diam, 10mm long</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>9 well plates 85X100 mm, 22mm o.d.x7mm deep</td>
<td>&nbsp;</td>
<td>CORNING</td>
<td>7220-85</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3516</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Menzel Glaser GmbH</td>
<td>798</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940-223</td>
<td>&nbsp;</td>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vectashield</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>FAL352350</td>
<td>&nbsp;</td>
<tr>
<td>1B1 antibody</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Traffic Jam</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Dorothea Godt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Vasa</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Ruth Lehmann</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti &beta;-Galactosidase</td>
<td>&nbsp;</td>
<td>Cappel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibodies</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dissection and staining of drosophila larval ovaries

多くの臓器は、胚発生時と成人期の間にメンテナンスや修理のための彼らの開発のための幹細胞に依存する。どのように幹細胞形態、およびそれらがどのようにその環境と相互作用を理解することは、開発、ホメオスタシスと疾患を理解するため、非常に重要です。 ショウジョウバエの卵巣は、彼らの体細胞の支持細胞（ニッチ）1、2の生殖系列幹細胞（GSCs）の相互作用のための有力なモデルを務めている。遺伝的にそれらを操作する能力に結合ニッチの既知の場所とGSCsは、研究者は幹細胞とそのニッチ3月12日の間の相互作用の様々な解明することができました。 GSCメンテナンスと分化の制御機構に関する豊富な情報にもかかわらず、比較的少ないがGSCsとその体細胞のニッチは、開発中に形成する方法については知られている。その細胞成分の端末フィラメントとキャップの細胞（図1）、第三幼虫齢13から17の間にフォームを含む約18体細胞ニッチ、。 GSCsは、始原生殖細胞（始原生殖細胞）に由来する。 PGCは初期の幼虫の段階で増殖するが、ニッチの形成に続いて始原生殖細胞のサブグループはGSCs 7、16、18、19になります。一緒に、体細胞のニッチ細胞とGSCsは、生物の生涯を通じて卵を生成する機能的なユニットを作る。 GSCユニットの形成に関する多くの質問が未回答のままである。このようなニッチと幹細胞の前駆体の前駆細胞間の調整、またはそれらがGSCsになると始原生殖細胞内の非対称性の生成などのプロセスは、最善の幼虫で検討することができます。しかし、幼虫の卵巣の開発の方法論の研究は、物理的に困難です。最初に、幼虫の卵巣は小さくなります。であっても後期幼生の段階で彼らは、全体で唯一の100μmのです。さらに、卵巣は透明で、白い脂肪体に埋め込まれています。ここでは、蛍光抗体で染色することによってこの後に続く三齢（LL3） ショウジョウバエの幼虫、から卵巣を分離するためのステップバイステップのプロトコルについて説明します。我々はそのような、卵巣の位置をシンクしていない組織を染色し、洗浄し、抗体が組織に浸透することを確認してなどの問題に対していくつかの技術的ソリューションを提供しています。このプロトコルは、以前の幼虫の段階にとだけでなく、幼虫の精巣に適用することができます。

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Larval Ovaries at JoVE.com

Dissection and Staining of Drosophila Larval Ovaries

開発時にどのようにニッチと幹細胞の形態は、実用的な意味を持つ重要な問題です。の<em&gt;ショウジョウバエ</em&gt;卵巣、生殖系列幹細胞とその細胞のニッチは、幼虫の発育中に形成する。このビデオでは、後期三齢（LL3）から女性の生殖腺を、解剖染色およびマウントする方法を示しています<em&gt;ショウジョウバエ</em&gt;幼虫。

の解剖と染色ショウジョウバエ幼虫卵巣

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

dissection-and-staining-of-drosophila-larval-ovaries

Research

JoVE Journal

Biology

Dissection and Staining of Drosophila Larval Ovaries

26.1K ビュー.  Weizmann Institute of Science. 開発時にどのようにニッチと幹細胞の形態は、実用的な意味を持つ重要な問題です。のショウジョウバエ卵巣、生殖系列幹細胞とその細胞のニッチは、幼虫の発育中に形成する。このビデオでは、後期三齢（LL3）から女性の生殖腺を、解剖染色およびマウントする方法を示していますショウジョウバエ幼虫。

ビデオ: Dissection and Staining of Drosophila Larval Ovaries

Dissection of Drosophila Ovaries

ショウジョウバエの卵巣の解剖

生物学

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ショウジョウバエ幼虫NMJの解剖

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

ショウジョウバエ幼虫NMJ免疫組織化学

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

大人からOenocytesの解剖キイロショウジョウバエ</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

後期胚および幼虫の超音波処理により促進免疫蛍光染色<em&gt;ショウジョウバエ</em&gt;組織<em&gt;その場で</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖や成虫の免疫染色から<em&gt;キイロショウジョウバエ</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖と実装<em&gt;ショウジョウバエ</em&gt;蛹アイディスク

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...