私たちは、私たちはクライオミクロトームを使用できるようにするため教授Eicheleに感謝。作業は、マックスプランクGesselschaftによって賄われていた。

1。準備<ol><li>たて3これは。、無水エタノールを組み合わせることにより、クロロホルム、それぞれの割合午前6時03分01秒の氷酢酸をカルノア固定液を準備するだけでなく、水没の襟のすべてのソリューションは、ガラスの染色瓶（推薦のための試薬 ​​の項を参照）に保管する必要があります。 </li><li>首輪の正しいサイズのアルミ箔のポケットを準備します。 </li><li> 2 X 40％エタノール、70％エタノール、2 X 100％エタノール、メチルベンゾエート（MB）、50/50 v / vのMBおよびパラフィン、2 Xのパラフィン：jarを染色では、次の溶液を調製。 60〜65℃にインキュベーターセットのMBとパラフィンの入った容器を置かない</li><li> 60〜65℃に箔のポケットに注いで温かいパラフィン</li></ol> 2。カラーでハエを修正<ol><li>その場のためのエントリポイントを見ることができる垂直位置でテープを使用して両眼の下襟を取り付けます4。 </li><li>麻酔は、二酸化炭素を使用して、または氷のブロックを使用して低体温を経由して飛ぶ。ハエを凍結しないように注意してください。 </li><li>鉗子を使用して、（刃と刃下の腹部の上に頭部と胸部）を正しい方向に向けて襟に羽と場所をつかんで、個々のハエを拾う。 10月20日ハエは、簡単に襟に収まる必要があります。 </li></ol>注：複数の遺伝子型を分析している場合は、カラー数と対応する遺伝子型をメモしておくことを忘れないでください。 3。 ショウジョウバエ胸部のパラフィン切片<ol><li>カルノア液に襟を再配置し、4℃で組織を固定° Cを一晩。 </li><li>固定後、エタノールの濃度を増加させて使用してサンプルを脱水する。 10分ごとに沈めるために室温で40％（2倍）、70％と100％（2回）エタノールに襟。 [次へ]をインキュベートの各30分間MBとMB +パラフィン溶液（1:1）でのカラーとは、60〜65で60分間ずつパラフィンの二つの変更℃で襟に潜入急速に箔のポケットに襟を再配置し、溶融（60〜65 ° C）パラフィンを詰めます。室温で入れ、パラフィンが（それは一晩おくのが最適です）ハードになることができます。必要なセクション（縦または横）の方向に応じて、カラーが異なる方向に箔のポケットに置くことができることに注意してください。 </li><li>箔から首輪をつけて乾燥したパラフィンブロックをアンラップし、静かにパラフィンブロックから襟を分離。鋭い刃またはメスの助けを借りて、慎重にフライの組織の周りから余分なパラフィンを切り取る。 </li><li>回転ミクロトーム7-10μmのセクションの手順でパラフィンブロックをカットし、カットの組織は37℃の水浴でフラットフロートすることができます。極スライドに組織切片を置き、一晩乾燥できます。これらのスライドは、組織構造を可視化するためにヘマトキシリン​​およびエオシン（図1A - D）、トルイジンブルー、アニリンブルーや他の金型と染色のためだけでなく、抗体染色のために使用することができます（図1E - E ``）。 </li></ol> 4。 ショウジョウバエ胸部の凍結切片<ol><li>パラフィン切片の場合と同様に、襟とファッションアルミ箔のポケットにハエを準備。凍結クーラーは、サンプルを調製するために必要とされる。クーラーは-60程度であることを確認してください° Cを、この温度に到達できるように、少しエタノールとドライアイスを使用してください。実験を開始する前に、時間はそれを冷却すると、気泡の形成を最小限にするために4℃の冷蔵庫に逆さまに低温埋め込み培地のボトル（組織- Tek社のOCT化合物）を置く。 </li><li>凍結クーラー内部の数分間、事前に冷却し、急速凍結包埋化合物でいっぱいにされている箔のポケットにハエと襟を再配置します。 30〜10分間、サンプルの凍結をしてみましょう。慎重に、クーラー内部に形成されたブロックをアンラップそっと埋め込みのブロックから襟を分離し、少なくとも一日は-20℃でそれを置く。 </li><li> 10月15日μmの切片厚を-15〜-18℃の間凍結ミクロトームで凍結した筋肉を切り取ります。偏スライド上に配置し、さらに処理を行う準備まで-20℃で保管。我々は、抗体染色の前に室温で10分間、4％ホルムアルデヒドPBS溶液中で組織を固定示唆している。 </li></ol> 5。 ショウジョウバエの筋肉の脂質の検出脂肪滴は、オイルレッドOは、シーバーとThummel 5から採用されたプロトコルを使用して凍結切片を染色で検出することができます。 <ol><li>固定後、オイルレッドOで3時間10分インキュベートを室温で染色するためのプロピレングリコール中で平衡化5分、のために水で2回スライドを洗浄する。その後、プロピレングリコール及びPBSで30分で5分間サンプルを2回洗浄する。 30％グリセロールでマウントします。 </li></ol> 6。代表的な結果： <img src="/files/ftp_upload/2438/2438fig1.jpg" alt="図1" /> 図Figure 1に。 Parrafin組み込みのセクション 間接飛翔筋のヘマトキシリン​​とエオシンで染色横（AB）と縦（CD）のセクション。とCは通常の構造化された筋肉を示しています。サイズと形態の筋肉の異常は、それぞれBとD（黒矢印）で表されます。 （EF）核染色法抗LamC、核膜のマーカーとDAPI、 ショウジョウバエの胸部のステンドグラスの横断面。正常の拡大図（赤矢印）と劣化（黄矢印）筋肉（F）。 GはLamCとDAPIで染色したショウジョウバエ腸管のセクションを表します。 <img src="/files/ftp_upload/2438/2438fig2.jpg" alt="図2" /> 図2。凍結切片 オイルレッドO、脂質滴のラベルで染色されたショウジョウバエの胸部のA.横断凍結切片。 間接飛翔筋のB.縦凍結切片は、抗- DG、筋肉筋鞘マーカーとDAPIで染色した。

<ol>
	<li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+anatomy+and+histology+of+the+imago+of+Drosophila+melanogaster.">The internal anatomy and histology of the imago of Drosophila melanogaster.</a> , (1950).</li><li>Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+muscle+and+neuronal+disorders+in+a+Drosophila+model+of+muscular+dystrophy.">Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy.</a> The EMBO journal. 26, 481-481 (2007).</li><li>Kucherenko, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modifier+screens+reveal+new+components+that+interact+with+the+Drosophila+dystroglycan-dystrophin+complex.">Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex.</a> PloS one. 3, e2418-e2418 (2008).</li><li>Puchtler, H., Waldrop, F. S., Conner, H. M., Terry, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carnoy+fixation:+practical+and+theoretical+considerations.">Carnoy fixation: practical and theoretical considerations.</a> Histochemie. 16, 361-36 (1968).</li><li>Ashburner, M. <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 - A Laboratory Manual. , (1989).</li><li>Sieber, M. H., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DHR96+nuclear+receptor+controls+triacylglycerol+homeostasis+in+Drosophila.">The DHR96 nuclear receptor controls triacylglycerol homeostasis in Drosophila.</a> Cell metabolism. 10, 481-481 (2009).</li></ol>

利害の衝突は宣言されません。

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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>Stainless steel collars</td>
<td>&nbsp;</td>
<td>self-made</td>
<td>&nbsp;</td>
<td>Specially constructed</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools GmbH</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Aluminum foil</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Blade or scalpel</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wheaton macro staining jar</td>
<td>&nbsp;</td>
<td>Wheaton Science Products </td>
<td>900200</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>&nbsp;</td>
<td>Model: Hyrax M25</td>
</tr>
<tr>
<td>Cryo-microtome</td>
<td>&nbsp;</td>
<td>Leica </td>
<td>&nbsp;</td>
<td>Model CM3050S</td>
</tr>
<tr>
<td>60-65&deg;C Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>Large enough to hold at least 4 staining jars</td>
</tr>
<tr>
<td>Freezing cooler with metal block</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>Store at -80&deg;C</td>
</tr>
<tr>
<td>Super-frost slides</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>9161155</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Any </td>
<td>&nbsp;</td>
<td>Recommend 24 X 40 mm</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>288306</td>
<td>Analytical grade </td>
</tr>
<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>100063</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>100983</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Methylbenzoate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M29908-500G</td>
<td>Analytical grade</td>
</tr>
<tr>
<td>Paraplast plus</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>76258</td>
<td>Paraffin</td>
</tr>
<tr>
<td>Tissue-Teck O.C.T. compound</td>
<td>&nbsp;</td>
<td>Sakura</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>16% formaldehyde, methanol free</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>18814</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G5150-1L</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

paraffin-embedded and frozen sections of drosophila adult muscles

ヒトの筋ジストロフィーとミオパシーの分子特性は、筋肉の病気と筋肉の仕様の遺伝学的解析の複雑さを明らかにした、モデルシステムの形成と機能は、筋肉の生理学に貴重な洞察を提供しています。そのため、筋肉の損傷を根底にある分子機構を同定し、特徴付けることが重要です。成人ショウジョウバエマルチファイバーの筋肉の構造は、脊椎動物の横紋筋1とショウジョウバエの遺伝学的従順さがそれをジストロフィー筋の形態を分析し、加齢成人で筋肉機能に影響を与えるプロセスが2便を運行する航空会社の特性に優れたシステムをした似ている。ここでは、 ショウジョウバエの胸部筋肉のパラフィン包埋して凍結切片を調製するための組織学的手法を提示する。これらの製剤は、古典的な組織学的染色で染色し、タンパク質を検出する色素で標識する組織を可能にし、特に凍結切片はそのまま筋肉の蛋白質の免疫組織化学的検出に最適です。これは、筋肉組織の構造、形態学的欠陥の同定、および筋肉/ ショウジョウバエの成体の筋におけるニューロン特異的タンパク質の発現パターンの検出の解析が可能になります。これらの技術はまた、少し他の身体部分の切片用に変更することができます。

Watch this Scientific Journal Video about Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles at JoVE.com

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

筋肉の損傷のメカニズムの同定は非常に重要です。ここでは、ショウジョウバエの胸部筋肉のパラフィン包埋して凍結切片を調製するための組織学的手法を提示する。これは筋肉の形態やタンパク質と他の筋肉の細胞成分の局在の分析を行うことができます。

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

paraffin-embedded-and-frozen-sections-of-drosophila-adult-muscles

Research

JoVE Journal

Biology

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

36.4K ビュー.  Max Planck Institute for Biophysical Chemistry. 筋肉の損傷のメカニズムの同定は非常に重要です。ここでは、ショウジョウバエの胸部筋肉のパラフィン包埋して凍結切片を調製するための組織学的手法を提示する。これは筋肉の形態やタンパク質と他の筋肉の細胞成分の局在の分析を行うことができます。

ビデオ: Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

アダルトショウジョウバエの脳から全細胞レコーディング

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

生物学

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

開発と大人の準備ショウジョウバエ</emライブイメージングのための>脳と網膜

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

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 ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

遺伝学的およびエピジェネティクス解析のためのパラフィン包埋材料からのDNA抽出

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

大人の準備ショウジョウバエ</emシンセクショニングおよび顕微鏡分析のための>アイズ

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

ホルマリン固定からプロテオミクスサンプル調製およびパラフィン包埋組織

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCRは、ホルマリン固定、パラフィン包埋された生検組織中のサイトメガロウイルスDNAの検出のための高感度かつ迅速な方法であり、

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

マウス乳腺の凍結切片の間接免疫

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

ホルマリン固定パラフィン包埋参照標準細胞株にBRAF V600E変異を検出するデジタルドロップレットPCRを採用

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

RNA及びDNA抽出の両方のためのホルマリン固定パラフィン包埋組織コアの調製

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...