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1。日1 始める前に： ハエの健全な人口は、標準的な培養のプロトコルを使用して維持する必要があります：卵レイと開発は第3齢幼虫がpupariationを開始放浪するまで一定温度での続行を許可するの3-4日後にボトルやバイアルから大人を削除します。幼虫/蛹の遷移がprepupae（0時間蛹殻形成- APFの後に考えられる）の形成によってマークされています。不動の蛹はその白の着色により、以前の蛹から、まだ彼らの細長い、丸みを帯びた形状と前部気門の突起によってpupariationを開始していない幼虫から区別される。 次のものが必要です。 <ul><li>蛹を収集するためのペイントブラシ</li><li>培養蛹のための湿気のあるチャンバー：ペトリ皿は、接液ペーパータオルで並んでと蛹の収集、遺伝子型または性別の種々の時点のマークが付けられたフィルターの紙で覆われ</li><li> 1Xリン酸は、サナギを洗浄するための緩衝食塩水（PBS） </li></ul>収集、培養およびステージング蛹<ol><li>接液絵筆を使用すると、静かに培養ボトル/バイアルから0hr APFのサナギを削除し、湿気のあるチャンバーの蓋に置いてください。 </li><li>絵筆と1X PBSを使用すると、そっと蛹のケースから破片を除去するためにサナギを洗う</li><li>必要に応じてソート性別によるサナギの場合。蛹の生殖巣原基は、男女をソートするために使用することができます。男性の生殖腺原基は容易に蛹クチクラを通して約2 / 3体5の長さがダウンして見られる半透明のディスクの横方向のペアです。女性の生殖腺原基は小さく、同じように簡単に識別されません。 </li><li>湿度の高い室で適切にラベルの位置にサナギを置き、一定温度に戻ります。適切な発達時点まで文化。 </li></ol> 2。 2日目：解剖、固定し、一次抗体のインキュベーション始める前に： 次のものが必要です。 <ul><li>解剖顕微鏡</li><li>手術用鉗子</li><li> 11番ブレードを外科用メス</li><li>二九もガラスのうつ病の皿（コーニングの製品＃7220〜85） <ul><li> 1X PBSで一皿の井戸を埋める：これは、切除した蛹を洗浄するための洗浄皿です。 </li><li>固定バッファで2皿の井戸を埋める</li></ul></li><li>抗体のインキュベーションのための湿気のある部屋。我々は、接液ペーパータオルを敷いたプラスチック製の密閉式サンドイッチボックスを使用してください。 </li><li>固定バッファ：1X PBS、4％パラホルムアルデヒド、0.2％デオキシコール酸（透過化用） </li><li>解剖プラットフォーム：片側に接着した両面テープの切れ端でクリア顕微鏡用スライド</li><li> 1X PBS </li><li> P100やP200ピペッター</li></ul>解剖<ol><li>鉗子を使用すると、静かに一度蛹つを削除し、テープの広い幅が直面している蛹の前端と解剖のプラットフォーム上に置きます。蛹は過度にぬれた場合は、最初にそれらはペーパータオルを使用して乾燥さ汚点。 </li><li>蛹が完全にテープに付着した前に、それらを配置するために絵筆を使用。 </li><li>空気乾燥にサナギを許可し、テープ（5-10分）に準拠して</li><li>外科用メスで二国間でそれぞれの蛹を解剖。これは最高の前から蛹の後端に、単一の急速なカットで達成されます。適切に行われれば、カットは前部と後部の両方気門のペアを二分されます。サンプルを洗浄し、洗浄液として一度に10以上の蛹を解剖するには、迅速に組織のタンパク質分解の損傷を防止するために必要です。 </li><li>絵筆を使って、テープからそれらを緩めるために、各解剖サナギに1X PBSを少量転送する。 </li></ol>クリーニング、固定および一次抗体のインキュベーション<ol start="6"><li>手術用鉗子のペアで前方に個々のさなぎの半分をつかんで、すぐに洗浄皿のウェルに、それを浸す。解剖のプラットフォームは、顕微鏡ステージ上で洗浄皿に置き換える必要があります。処理が完了し、サンプルがマウントするための準備が整うまでは蛹のケースは、サンプルのままにしておく必要があります。 </li><li>まだ蛹の半分をつかんで軽く腹部の内部組織を洗い流すためにピペッターを使用してください。洗浄中に過剰な圧力は、上皮細胞の喪失につながることができます。 </li><li>直ちに、室温で固定バッファにきれいな蛹の半分を譲渡し、同様に残りの蛹の半分を処理。練習すれば、20の解剖と洗濯蛹の半分は5分未満を取る必要があります。 </li><li>蛹が1（1）時間室温で固定バッファでインキュベートすることができます。 </li><li> 1X PBSで固定サナギ3倍、5分を洗い流す</li><li>サンプルはすぐに、免疫組織化学のために処理されるか、または3ヶ月間の細胞またはエピトープ反応性を失うことなく-20℃で100％エタノールに格納されることがあります。 </li><li>サンプルを保存するために、希釈からサンプルを平衡化PBSのシリーズ：前に100％エタノールに移し、各希釈で20分間室温でエタノール（3:1、1:1、1:3）。 </li><li>サンプルは、免疫組織化学のために処理する前に、逆一連のPBSで再平衡にする必要があります。 </li><li>一次抗体を添加する前の1（1）時間のために1 × PBS（2％ウシ血清アルブミンを補充）でサンプルをブロックする。 </li><li> 4℃で一晩1X PBSで適当な濃度に希釈した一次抗体°揺動することなくCでインキュベートする。 </li></ol> 3。 3日目：二次抗体のインキュベーション、取り付けとイメージング始める前に： 次のものが必要です。 <ul><li> 2つの手術鉗子</li><li>マウントメディア（グリセロール、Vectashield [ベクタ​​ー研究所]、またはSlowfadeゴールド[インビトロジェン]） </li><li>うつ病も顕微鏡スライド（0.8 mm）の</li><li>カバーグラス（24 × 40 mm）を</li></ul>二次抗体のインキュベーションおよび取付け： <ol><li> 1 × PBSで各サンプルを3倍の10分を洗う</li><li>二次抗体を添加する前の1（1）時間のために1 × PBS（2％ウシ血清アルブミンを補充）でサンプルをブロックする。 </li><li> 3（3）時間のためにロッキングすることなく、暗所で室温で1 × PBSで適当な濃度に希釈した二次抗体とインキュベートする。 </li><li> 1 × PBSで各サンプルを3倍の10分を洗う</li><li>該当する場合は、対比蛹（我々はDAPIで核を染色例[4&#39;、6 - ジアミジノ-2 - フェニル] 10分間PBSで希釈）。 </li><li>マウントする前に適切なメディアでのサンプル（30分の最小値）を平衡化させます。 </li><li>サンプルのスライドを準備します。蛹の形態は、平滑な取付を防ぎます。サンプルは、イメージングのためのうつ病のスライド（ドロップスライド）のウェルにマウントされます。場所もうつ病の取り付けメディアの75 - 100ul。いくつかのサンプルは、単一のスライドにマウントすることができます。 </li><li>蛹の場合は、マウントする前にサンプルから除去する必要があります。前方に内部蛹の膜を把握しないようにしている個々の蛹のケースをつかみます。 </li><li>鉗子の2番目のペアで軽く頭で内部蛹の膜を把握し、蛹のケースから取り外します。 </li><li>すぐにもうつ病にサンプルを転送し、追加のサンプルの処理を続行する。 </li><li>うつ病でも外側面を上に向けてサンプルに均一プローブまたは鉗子の位置を使用する。時折気泡がプローブを削除することができます。 </li><li>ゆっくりと気泡を導入しないように注意しながら試料の上にカバースリップを下げる</li></ol> 4。代表的な結果： サンプルは、このプロトコルは、成人の腹部の総形態を保持用いて調製。画像のスタックは、腹部のトポロジを調査するために適用することができる二次元画像または3 - Dレンダリングを生成すると予想されることがあります。 <img src="/files/ftp_upload/3139/3139fig1.jpg" alt="図1"/> 図1。 。 ショウジョウバエ蛹翼のない蛋白質と縁をギザギザにした発現（EN - GAL4：UAS - GFP） におけるセグメンテーションの遺伝子産物は、：と抗GFPマウス抗- WG（アイオワハイブリドーマバンク4D4）と（APF）蛹殻の形成後26時間で可視化した。 10倍の倍率は、前方は左だと背が最高です。 NucleiiはDAPIで対比染色した。 約50枚（スライス間のΔZは 2.5μmである）のスタックが予測された。

<ol>
	<li>Kopp, A., Duncan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anteroposterior+patterning+in+adult+abdominal+segments+of+Drosophila.">Anteroposterior patterning in adult abdominal segments of Drosophila.</a> Dev. Biol. 242, 15-30 (2002).</li><li>Ninov, N., Chiarelli, D. A., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+and+intrinsic+mechanisms+directing+epithelial+cell+sheet+replacement+during+Drosophila+metamorphosis.">Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis.</a> Development. 134, 367-379 (2007).</li><li>Bischoff, M., Cseresnyes, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+rearrangements,+cell+divisions+and+cell+death+in+a+migrating+epithelial+sheet+in+the+abdomen+of+Drosophila.">Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.</a> Development. 136, 2403-2411 (2009).</li><li>Matsuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+Morphology+and+evolution+of+the+insect+abdomen.">in Morphology and evolution of the insect abdomen.</a> , (1976).</li><li>Kerkis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+of+the+Gonads+in+Drosophila+Melanogaster.">The Growth of the Gonads in Drosophila Melanogaster.</a> Genetics. 16, 212-224 (1931).</li></ol>

利害の衝突は宣言されません。

このビデオで紹介するテクニックは、発達時のポイントの様々なショウジョウバエ蛹を準備するために使用することができます。 32時間に24時間の期間APF中に処理サナギは、APF上皮からの細胞の損失に最もなりやすいです。拡張インキュベーションステップの間に界面活性剤の使用は、（そのようなトリトンX - 100とTween - 20）細胞の損失の可能性が高く、したがって、推奨されません。むしろ、デオキシコール酸は、最初の固定ステップの間に洗浄剤として使用されます。後続のすべての手順は、洗剤なしで1X PBSで実行されます。さらに、拡張されたインキュベーションステップの間に試料を揺動する細胞の損失を増加させ、回避する必要があります。 サンプルは共焦点顕微鏡技術を用いて画像化することができます。しかし、説明したように処理されたショウジョウバエ蛹はまた共焦点技術に匹敵する画質をもたらす構造化照明顕微鏡システムを用いて画像化することができます。

<ul><li>解剖顕微鏡</li><li>手術用鉗子</li><li> 11番ブレードを外科用メス</li><li>九ウェルガラスのうつ病の皿（コーニング製品＃7220〜85） </li><li>培養蛹のための湿気のあるチャンバー</li><li> 1Xのリン酸緩衝生理食塩水（PBS） </li><li> ＃11外科用メス</li><li>両面テープ</li><li>固定バッファ：1X PBS、4％パラホルムアルデヒド、0.2％デオキシコール酸（透過化用） </li><li> P100やP200ピペッター</li><li>うつ病も顕微鏡スライド（0.8 mm）の</li></ul>

drosophila pupal abdomen immunohistochemistry

ショウジョウバエの蛹の腹部には、上皮形態形成と1-3性的二形性形態の開発の研究のための確立されたモデルシステムです。 （25℃）で約96時間にわたって蛹化、中に、成虫の細胞の増殖集団は、大人の腹部のセグメントを生成するために幼虫の表皮を交換してください。これらの成虫の細胞は、胚形成の間に生まれ、幼虫の各腹部セグメントにおける組織原細胞の巣の外側のペアとして存在する。組織原細胞の巣の4つのペアは、大人の背側クチクラ（前部と後部の背巣）、腹側クチクラ（腹側の巣）と、各セグメント（気門巣）4に関連付けられている気門を生じさせる。 puparation時には、これらの二倍体細胞は、（より大きい倍数体幼生表皮細胞- LECSからサイズによって区別できる）の増殖、遊走およびLECの交換のステレオタイプのプロセスを開始します。様々な分子と遺伝学的ツールは、成人の腹部の形態形成に関与する遺伝的経路の寄与を調査するために用いることができる。究極の大人の表現型は、通常、成人の腹部キューティクルの以下の解剖を分析している。しかし、基盤となる分子過程の研究は、固有の課題を提示蛹上皮の免疫組織化学的分析を、必要とする。時間的に動的な形態形成と二つの異なる上皮集団（幼虫と成虫）の相互作用は、解剖やその後の処理中に、過剰な細胞の損失を受けやすい脆弱な組織を生成する。我々は解剖、固定、マウントと共焦点または標準的な蛍光顕微鏡に適した一貫性のある高品質のサンプルを生成する免疫組織化学的研究のためのショウジョウバエ蛹のabdominem上皮のイメージングの方法を開発した。

Watch this Scientific Journal Video about Drosophila Pupal Abdomen Immunohistochemistry at JoVE.com

Drosophila Pupal Abdomen Immunohistochemistry

の抗体染色<em&gt;ショウジョウバエ</em&gt;蛹は成人腹部発生遺伝学の遺伝学的解析を強化することができます。我々は段階の解剖、固定し、抗体染色のために我々のプロトコルを提示<em&gt;ショウジョウバエ</em&gt;蛹の腹部。

ショウジョウバエの蛹の腹部の免疫組織化学

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

drosophila-pupal-abdomen-immunohistochemistry

Research

JoVE Journal

Biology

Drosophila Pupal Abdomen Immunohistochemistry

16.1K ビュー.  University of Alabama. の抗体染色ショウジョウバエ蛹は成人腹部発生遺伝学の遺伝学的解析を強化することができます。我々は段階の解剖、固定し、抗体染色のために我々のプロトコルを提示ショウジョウバエ蛹の腹部。

ビデオ: Drosophila Pupal Abdomen Immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

免疫組織化学のためのスライス標本の薄いセクショニング

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

生物学

In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.

In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.

リボプローブを用いたバタフライ蛹ウィングスのためのその場プロトコルで

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫組織化学：ベクターLabsからベクタステインABCキットを用いたパラフィン切片

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

蛍光抗体を用いたマウス表皮のパラフィン切片で免疫組織化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

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

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

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

ヒトタンパ​​ク質アトラス内の組織マイクロアレイ、免疫組織化学染色とデジタル化の生産

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

イメージングと免疫組織化学のための網膜ホールマウントの準備のために最適化されたプロトコル

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

の蛹ケースを画像化<em&gt;キイロショウジョウバエ</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖、イメージング、および薬理学的治療： ショウジョウバエ蛹精巣や独身男性生殖ライン嚢胞のex vivo培養

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

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 ...