Name: methods to examine the lymph gland and hemocytes in drosophila larvae
Uploaded: 2016-11-28T14:00:00.000+00:00
Duration: 11 min 49 s
Description: Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

We thank Matthew O'Connell, Maryam Jahanshahi, and Andreas Jenny for assistance. We thank Istv&#225;n And&#243; for plasmatocyte-specific antibodies, Utpal Banerjee for dome-meso-EBFP2 flies, Julian Martinez-Agosto for antp&gt;GFP flies, and Michael O'Connor for ptth and ptth&gt;grim flies. These methods were developed with support by the Kimmel Foundation, the Leukemia &amp; Lymphoma Society, NIH/NCI R01CA140451, NSF 1257939, DOD/NFRP W81XWH-14-1-0059, and NIH/NCI T32CA078207.

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著者は、彼らが何の競合や金融利害関係を持たないことを宣言します。

遺伝的または環境の変化に応じ、ここで説明した4つの方法は、シグナル伝達、生存、増殖、および分化のような造血中に別個のプロセスを分析するために、個別に、または組み合わせて使用することができるショウジョウバエ造血は、動的なプロセスです。動物あたりの血球数が35増加し、リンパ腺の構造と遺伝子発現は、開発中に32を変更します 。これらの?...

血液疾患における造血系の開発とその誤動作の座標転写因子およびシグナル伝達経路を調節する複雑なメカニズムはよくわかっていないままです。これらの転写因子およびシグナル伝達経路、ならびにそれらの調節は、非常にショウジョウバエおよび哺乳動物の造血1~5の間で保存されています。したがってショウジョウバエ造血系は、造血及び下地血液疾患を制御する分子機構を定義するための優れた遺伝的モデルを表します。 哺乳類と同様に、 ショウジョウバエは、造血の空間的および時間的に別個の相では、血球と呼ばれる、血液細胞を生成します。伝統的に、 ショウジョウバエの造血は、胚中胚葉にし、幼虫のリンパ腺に段階に限定されると考えられていました。最近の研究は、造血はまた、幼虫の無茎性のCLUで発生するという証拠を提供しますstersと6-8腹部大人インチplasmatocytesと液晶セル：すべての造血相は、成熟血球の2種類を生成します。 Plasmatocytesは食作用、先天性免疫、および創傷治癒に関与するマクロファージ様細胞です。クリスタル細胞がメラニン化のために必要なプロphenoloxidases、昆虫の免疫応答および創傷治癒に使用される反応が含まれています。幼虫の造血は、このような寄生蜂感染9,10などの特定の免疫の課題に応じて、lamellocyteと呼ばれる第三の成熟した血球タイプを、生成することができます。 Lamellocytesは、 ショウジョウバエの幼虫に敷設されたスズメバチの卵をカプセル化し、中和するために、plasmatocytesと液晶セルと組み合わせて、機能大、接着細胞です。寄生虫感染の非存在下で、lamellocytes野生型幼虫では見られません。メラニン質量はメラニン、カプセル化されたスズメバチの卵に似ています。多くの変異体ショウジョウバエ株は、寄生虫感染の非存在下での黒色腫瘤を開発します。 lamelloの存在球および/またはメラニンの塊は、造血異常を示すことができます。実際には、黒色質量表現型は、造血11-14に関与する遺伝子および経路を同定するために使用されています。 幼虫の造血系は、最も広く今日までに研究されています。それは、血リンパ中を循環血球、キューティクルの下にパターン化さ固着血球クラスター、およびリンパ腺に存在する血球から構成されています。リンパ腺は背容器に取り付けられた二国間のローブのシリーズです。リンパ腺の各一次ローブは、3つの主要な領域に分割されます。最も外側のゾーンは、皮質ゾーンとして知られており、成熟血球が含まれます。最も内側のゾーンは、髄質ゾーンと呼ばれ、静止血球前駆体から構成されています。第三ゾーン、後部のシグナリングセンターでは、幹細胞様ニッチとして機能リンパ腺の基部にある細胞の小グループです。初期の研究は、Notch 15-18のための重要な機能を確立しました</suP&gt;、ヘッジホッグ19,20、JAK-STAT 18、および無翅21の活動は、幼虫のリンパ腺の開発を規制します。より最近の研究は、BMP 22、FGF-RAS 23、およびカバ24,25シグナリングはまた幼虫のリンパ腺内で機能することを実証しました。 ここで説明四幼虫造血アッセイ1を説明する）3） インビボでの液晶セルを可視化する、2）免疫組織化学のために循環血球を単離し、固定し、単位体積あたりの細胞数として定義血球濃度が、循環、及び4）、解剖固定、実装測定免疫組織化学のための腺リンパ。これらのアッセイは、幼虫の造血系内シグナル伝達経路の機能および規則を評価するために、造血読み出しとして使用することができます。これらのメソッドは、フィールドで以前に使用されてきたが、これらのアッセイの視覚的なドキュメントは、ごく最近8,26-30を開始しました。ここで引用したいくつかの刊行物は、ヘルプです同様の方法および造血マーカー26,31-33を説明FUL資源。さらに、TROLとバイキングは、リンパ腺の基底膜の有用なマーカーです。

<table><tbody><tr><td>PBS tablets</td><td>MP Biomedicals</td><td>2810305</td><td></td></tr><tr><td>dissecting dish</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>microcentrifuge tube</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>silicone dissecting pad, made from Sylgard 184 kit</td><td>Krayden (distributed through Fisher)</td><td>NC9644388 (Fisher catalog number)</td><td>Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.</td></tr><tr><td>stereomicroscope</td><td>Morrell Instruments (Nikon distributor)</td><td>mna42000, mma36300</td><td>Nikon models SMZ1000 and SMZ645</td></tr><tr><td>tissue wipe</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>forceps</td><td>Electron Microscopy Sciences</td><td>72700-DZ</td><td></td></tr><tr><td>p200 pipette</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>hemocytometer</td><td>Hausser Scientific</td><td>3200</td><td></td></tr><tr><td>trypan blue stain</td><td>Life Technologies</td><td>T10282</td><td></td></tr><tr><td>formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>Triton</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tween 20</td><td>Fisher</td><td>BP337-500</td><td></td></tr><tr><td>bovine serum albumin</td><td>Rocky Mountain Biologicals</td><td>BSA-BSH-01K</td><td></td></tr><tr><td>normal goat serum</td><td>Sigma</td><td>G9023-10ML</td><td></td></tr><tr><td>normal donkey serum</td><td>Sigma</td><td>D9663-10ML</td><td></td></tr><tr><td>200 proof ethanol</td><td>VWR</td><td>V1001</td><td></td></tr><tr><td>N-propyl gallate</td><td>MP Biomedicals</td><td>102747</td><td></td></tr><tr><td>glycerol</td><td>VWR</td><td>EM-4750</td><td></td></tr><tr><td>DAPI (4&amp;rsquo;,6-diamidino-2-phenylindole)</td><td>Fisher</td><td>62248</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>351146</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>351143</td><td></td></tr><tr><td>microscope cover glass, 22 mm square</td><td>Fisher</td><td>12-544-10</td><td></td></tr><tr><td>microscope cover glass, 18 mm circular</td><td>Fisher</td><td>12-545-100</td><td></td></tr><tr><td>glass microscope slides</td><td>Fisher</td><td>22-034-980</td><td></td></tr><tr><td>thermal cycler</td><td>Eppendorf</td><td>E950010037</td><td>Mastercycler EP Gradient S</td></tr><tr><td>PCR tubes</td><td>USA Scientific</td><td>1402-2700</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>351147</td><td></td></tr><tr><td>disposable transfer pipet</td><td>Fisher</td><td>13-711-9AM</td><td></td></tr><tr><td>fluorescence microscope</td><td>Zeiss</td><td></td><td>Axio Imager.Z1</td></tr></tbody></table>

methods to examine the lymph gland and hemocytes in drosophila larvae

多くの類似点は、 ショウジョウバエ、哺乳動物の適応免疫を特徴付けるリンパ系統を欠いていても、 ショウジョウバエおよび哺乳動物の造血系の間に存在し、ショウジョウバエおよび哺乳動物の造血は、いくつかの血液細胞系統を生成するために空間的および時間的に異なる段階で起こります。両方のシステムが成熟した系統を展開したり交換すると、血液前駆細胞の貯水池を維持します。造血系は、 ショウジョウバエや哺乳類が応答すると免疫の課題に適応することができます。重要なのは、生成、保守、および造血系の機能を制御する転写調節因子およびシグナル伝達経路は、哺乳動物へのハエから保存されています。これらの類似性は、 ショウジョウバエは遺伝的モデル造血開発および疾患に使用することができます。 ここでは詳細なアッセイは、 ショウジョウバエの幼虫の造血系を検討しました。に特に、我々は、血液細胞数および濃度を測定生体内で特定の成熟した系統を視覚化し、循環中および造血器官に血液細胞の免疫組織化学法を実行するための方法を概説します。これらのアッセイは、遺伝子発現の変化及びシグナリング、生存、増殖、および分化を含む細胞プロセスを明らかにすることができるし、造血に関する質問の様々な調査するために使用することができます。 ショウジョウバエで利用可能な遺伝子ツールと組み合わせて、これらのアッセイは、定義された遺伝的改変の際に造血系を評価するために使用することができます。具体的にここで説明されていないが、これらのアッセイは、造血系の、そのような感染症や食事などの環境変化の影響を調べるために使用できます。

循環血球濃度血球数は、幼虫の発育35全体で増加します。この方法は、血球数と濃度の違いを検出することを説明するために、関係なく、生物学的原因の、私たちは、遅延および非遅延幼虫の血球濃度を測定しました。 PTTH-産生ニューロン（PTTH&gt;厳しい）の遺伝的アブレーションによる前胸腺刺激ホルモン（PTTH）?...

Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

ショウジョウバエおよび哺乳動物の造血系は、 ショウジョウバエの造血を研究するための魅力的な遺伝モデル作り、多くの共通の特徴を共有します。ここでは、免疫組織化学のための主要な幼虫の造血器官の解剖と取り付けを示しています。また、循環血球と固着液晶セルを含む様々な幼虫の造血コンパートメントをアッセイする方法を記載しています。

リンパ腺や血球中を調べるための方法<em&gt;ショウジョウバエ</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

methods-to-examine-the-lymph-gland-and-hemocytes-in-drosophila-larvae

Research

JoVE Journal

Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

12.8K ビュー.  The Icahn School of Medicine at Mount Sinai.  ショウジョウバエおよび哺乳動物の造血系は、 ショウジョウバエの造血を研究するための魅力的な遺伝モデル作り、多くの共通の特徴を共有します。ここでは、免疫組織化学のための主要な幼虫の造血器官の解剖と取り付けを示しています。また、循環血球と固着液晶セルを含む様々な幼虫の造血コンパートメントをアッセイする方法を記...

ビデオ: Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

の血液細胞集団のアッセイ<em&gt;キイロショウジョウバエ</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

発生生物学

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

免疫・感染学

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

幼虫給電回路の機能解析ショウジョウバエ</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

神経科学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

のライブイメージングキイロショウジョウバエ胚性血球移行

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

生物学

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

細菌感染や解析のためのキイロショウジョウバエ幼虫血球の抽出

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

インビボファゴサイトーシスアッセイを用いた成人ショウジョウバエのメラノガステル血球の年齢特異的食細胞能力の評価

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Drosophila Larval IHC

Immunohistochemistry (IHC) is a technique used to visualize the presence and location of proteins within tissues. Drosophila larvae are particularly amenable to IHC because of the ease with which they can be processed for staining. Additionally, the larvae are transparent, meaning that some tissues can be visualized without the need for dissection.
In IHC, proteins are ultimately detected with antibodies that specifically bind to &ldquo;epitopes&rdquo; within the protein of interest. In order to preserve these epitopes, tissues must be fixed prior to staining. Furthermore, in order for antibodies to penetrate membranes, cells must be permeabilized with detergents. This video article offers a detailed view of the reagents, tools, and procedures necessary for the staining of dissected larval tissues, including fixation, blocking, and staining steps. Also featured is a demonstration of tissue mounting techniques for fluorescence microscopy. Finally, examples of the broad range of applications for these techniques (and some variations upon them) are provided.

Drosophila melanogaster Larval Immunohistochemistry IHC

キイロショウジョウバエ幼虫を用いた免疫組織化学的検討

Immunohistochemistry (IHC) is a technique used to visualize the presence and location of proteins within tissues. Drosophila larvae are particularly ...

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基礎生物学

生物学Ⅰ：酵母、ショウジョウバエ、線虫

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

キューティクルの混乱:ショウジョウバエの幼虫から血リンパを採取する方法

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
The Drosophila ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster（ショウジョウバエ）

幼虫

Lipid Droplet Staining of Larval Drosophila Oenocytes: A Technique for Assessment of Lipid Droplet Accumulation Under Normal and Stressed Condition Using BODIPY-Based Dye

Source: Wei, C., et al., <a href="https://www.jove.com/v/60606">Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae.</a> J. Vis. Exp. (2019).
This video demonstrates lipid droplet staining in Drosophila larval oenocytes - hepatocyte-like cells - using the lipophilic fluorescent BODIPY based dye. These cells act as a model for studying lipid metabolism during development and in response to environmental stress such as starvation.

ショウジョウバエ幼虫の油滴染色:BODIPYベース色素を用いた正常条件およびストレス条件下での脂肪滴蓄積評価法

Source: Wei, C., et al., Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae. J. Vis. Exp. (2019).
This video demonstrates lipid ...

生物学的手法

染色技術

細胞染色

Larval Lymph Gland Dissection: Isolating the Drosophila Hemocyte-Producing Organ

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
This video describes the Drosophila lymph gland&#8212;the primary site of hematopoiesis in the fly larva&#8212;and shows an example protocol to dissect and mount the organ for immunohistochemistry and fluorescent imaging.

幼虫リンパ腺解剖:ショウジョウバエ血球産生器官の分離

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
This video describes ...

リンパ腺や血球中を調べるための方法

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幼虫給電回路の機能解析ショウジョウバエ