SG는 버팔로의 뉴욕 주립 대학에서와 존 R. Oishei 재단 기금에 의해 지원됩니다.

<html> 1. 시약의 준비 <ol><li> 100mM 파이프, 2mM EGTA, 1 MM MgSO 4를 사용 PEMFA 버퍼를 준비합니다. 7.0 버퍼의 산도를 조정합니다. PEMFA 버퍼는 필터 소독과 4 ° C.에 저장됩니다 </li><li> 중 1ml를 추가하여 PBT를 준비 트리톤 X - 100 100ml 1X PBS (인산이 살린 버퍼) (산도 7.0). 4 스토어 ° C. </li><li> 1X PBS (산도 7.0)에 0.01 % NaAcide와 5% BSA를 (소 혈청 알부민) 준비합니다. </li><li> 0.7 % NaCl 및 0.03 % 탈이온수에서 트리톤 X - 100을 사용하여 생리 식염수를 준비합니다. </li><li> 한 부분은 37 % 포름 알데히드 4 개 부품 PEMFA 버퍼를 결합하여 정착액을 준비합니다. </li></ol> 2. 태아 수집 <ol><li> 한천 먹이 플레이트와 어댑터를 포함한 플라스틱 병 속에&gt; 100 파리를 배치하여 관심 Drosophila 변형에 케이지를 준비합니다. </li><li> 파리가 케이지에 acclimated하고 그들의 계란을 보유하지 않게, 접시마다 8-12시간을 변경, 72 시간 동안 실온에서 어둠 속에 파리의 새장을 둡니다. </li><li> 실험은 12 시간 동안 배아를 수집하십시오. 컨테이너에서 페트리 접시를 제거하고 교체합니다. 삭제 페트리 접시를 가지고 생리 식염수와 깨끗한 페인트 브러쉬를 사용하여 요리에서 배아를 씻으십시오. </li><li> 훌륭한 체에서 배아를 수집합니다. 초과 누룩을 제거하는 호수와 린스. </li><li> 주걱을 사용하면 부드럽게 배아를 선택하고 모자와 작은 유리관에서 그들을 놓으십시오. 생리 식염수의 1ml에 대해 추가합니다. </li></ol> 3. Chorion 및 Vitelline 점막의 제거 및 고정 <ol><li> 배아의 수집에 따라 50 % 헵탄 50 %의 정착액 결합하여 헵탄 - 수정 솔루션을 준비합니다. 솔루션은 상단과 하단에 정착액에 헵탄와 계층 것입니다. </li><li> 유리관에 5 분 동안 생리 식염수에서 그들을 잠복기로 배아를 씻으십시오. 파스퇴르 피펫을 사용하여 여분의 호수를 제거합니다. </li><li> 탈이온수와 배아를 씻어. </li><li> 3 분 50 % 표백제에서 그들을 잠복기로 배아를 Dechorionate. </li><li> 탈이온수로 철저하게 배아를 씻어. </li><li> 부드러운 흔들림과 헵탄 - 수정 솔루션 30 분 동안 그들을 잠복기로 배아를 수정. </li><li> 파스퇴르 피펫을 사용하면 메탄올이 튜브에서 헵탄를 떠나있는 정착액 (하단 레이어)를 대체합니다. vitelline 막을 제거하기 위해 적극적으로 흔들어. devitellinized 배아는 튜브의 바닥에 싱크 것입니다. </li><li> 파스퇴르 피펫을 사용하여 새 유리관에서 배아를 수집합니다. 태아에게 메탄올이있는 5 번 린스, 각 시간 5 분 동안 태아를 품어. </li></ol> 4. 항체 스테 이닝, 마운트, 그리고 시각화 <ol><li> 50% PBT 솔루션과 5 분 50 % 메탄올의 혼합물에서 배아를 Rehydrate. 그렇다면 5 분 백퍼센트 PBT 솔루션 rehydrate. </li><li> 4 1 시간 0.01 % NaAcide 솔루션 ° C.와 함께 5 % BSA에서 그들을 잠복기로 배아를 차단 </li><li> 배아 지금 항체 염색법에 대한 준비가되어 있습니다. 0.01 % NaAcide 솔루션 5 % BSA를 사용하여 적절한 농도로 diluting하여 기본 항체를 준비합니다. 우리는 시스테인 문자열 단백질 (CSP), 기공을 갖는 시냅스 단백질과 표면 glycoproteins와 라벨 모든 뉴런에 존재의 연결을 특정 - 탄수화물 잔기를 인식 방지 HRP 또는 말 무 퍼옥시데이즈 항체, 대한 항체를 사용합니다. </li><li> 4 밤새 배아 ° C 기본 항체 솔루션 인치를 품어 </li><li> 다음 날, 시간의 과정을 통해 PBT와 배아 10 번 씻으십시오. </li><li> 적절한 농도를 diluting하여 이차 항체 솔루션을 준비합니다. 우리는 1:200의 농도에 알렉사 차 항체를 사용합니다. </li><li> 4 두 시간 동안 태아를 품어 ° C 보조 항체 솔루션 인치 </li><li> 시간의 과정을 통해 태아에게 PBT 10 번 씻으십시오. </li><li> Vectashield 장착 매체 하룻밤 배아를 품어. </li><li> 유리 슬라이드에있는 배아를 탑재하고 매니큐어로 커버 슬립을 봉쇄. 형광 현미경을 사용하여 배아를 시각화. </li></ol> 5. 대표 결과 <img src="/files/ftp_upload/2150/2150fig1a.jpg" alt="그림 1" /> CSP <img src="/files/ftp_upload/2150/2150fig1b.jpg" alt="그림 1" /> HRP - FITC <img src="/files/ftp_upload/2150/2150fig1c.jpg" alt="그림 1" /> 병합 그림 1 : CSP (적색)와 HRP (녹색)에 대한 항체를 사용하여 스테이지 16-17에서 배아 CNS를 보여주는 대표 이미지. 접합면의 별개의 얼룩을 확인합니다. <img src="/files/ftp_upload/2150/2150fig2a.jpg" alt="그림 2" /> CSP <img src="/files/ftp_upload/2150/2150fig2b.jpg" alt="그림 2" /> HRP - FITC &quot;/&gt; 병합 그림 2 : CSP (적색)와 HRP (녹색)에 대한 항체를 사용하여 스테이지 16-17에서 배아 PNS를 보여주는 대표 이미지. 주변 뉴런의 반복 패턴을합니다.

<ol>
	<li>Bodmer, R., Barbel, S., Sheperd, S., Jack, J. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+sensory+organs+by+mutations+of+the+cut+locus+of+D.+melanogaster.">Transformation of sensory organs by mutations of the cut locus of D. melanogaster.</a> Cell. 51, 293-307 (1987).</li><li>Bodmer, R., Carretto, R., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+of+the+peripheral+nervous+system+in+Drosophila+embryos:+DNA+replication+patterns+and+cell+lineages.">Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages.</a> Neuron. 3, 21-32 (1989).</li><li>Campos-Ortega, J. A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Embryonic Development of Drosophila melanogaster. , (1985).</li><li>Carroll, S. B., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+the+fushi+tarazu+protein+during+Drosophila+embryogenesis.">Localization of the fushi tarazu protein during Drosophila embryogenesis.</a> Cell. 43, 47-57 (1985).</li><li>Dambly-Chaudi&#232;re, C., Ghysen, A., Jan, Y. N., Jan, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+connections+between+imaginal+discs+in+Drosophila.">Muscle connections between imaginal discs in Drosophila.</a> Developmental Biology. 113, 288-294 (1986).</li></ol>

관심 없음 충돌 선언하지 않습니다.

Drosophila의 배아가의 연결을 개발하는 동안 세포와 분자 변화를 조사위한 강력한 시스템입니다. 이 프로토콜에서는 immunohistochemistry에 대한 전체 마운트 배아를 준비하는 방법을 증명하고있다. 우리는 캐롤과 스콧, 1985 년 설명된 프로토콜에서이 프로토콜을 적응있다. 이 프로토콜은 다른 항체의 연결을 테스트하는 적응 될 수 있지만, 항체의 최적화 할 수 있습니다. 또한, 배아 PNS와 CNS의 발달 결함 다른 돌연변이 라인에서 배아를 비교하여 쉽게 시각하실 수 있습니다. 우리는 성공적으로 모터 단백질 돌연변이 들고 태아의 PNS와 CNS를 시각화하기 위해이 프로토콜을 사용하고 있습니다. 높은 배율에서 우리는 또한 배아 PNS와 CNS 내에서 axonal 트랙을 평가했습니다.

<table><tbody><tr><td>37% formaldehyde</td><td>Fisher Scientific</td><td>BP531-25</td><td></td></tr><tr><td>CSP</td><td>Developmental Studies Hybridoma Bank</td><td></td><td></td></tr><tr><td>HRP-FITC</td><td>Jackson ImmunoResearch</td><td>323-095-021</td><td></td></tr><tr><td>Alexa Fluor 568 Goat Anti-Mouse IgG</td><td>Invitrogen</td><td>A-11004</td><td></td></tr><tr><td>Vectashield Mounting Medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr></tbody></table>

visualization of the embryonic nervous system in whole-mount drosophila embryos

Drosophila의 배아가의 연결을 발전의 세포 및 분자 기초를 조사하는 매력적인 모델 시스템입니다. 여기의 연결을 단백질에 항체를 사용하여 Drosophila 배아 신경 시스템의 시각화를위한 절차를 설명합니다. 전체 배아 주변 신경 및 중추 신경 시스템이 잘 개별 세포 수준 (..; Bodmer 외, 1987;. Dambly - 쇼디에르 외, 1986 Bodmer 외, 1989)에서 특징 때문에, 이러한 시스템에 어떤 aberrations 수 쉽게 다른 단백질의 연결을하기 위해 항체를 사용하여 식별됩니다. 개발 배아는 배아는 시각화에 대한 적절한 발달 단계에 있는지 확인하기 위해 특정 시간에 수집하고 있습니다. 수집 후, 배 (胚)의 바깥쪽 레이어, chorion 막과 배아를 둘러싼 vitelline 봉투가 고정되기 전에 제거됩니다. 배아는 다음의 연결 항체와 incubated하고 찬란 레이블 차 항체를 사용하여 시각입니다. 단계에서 배아가 12-17 배아 신경 시스템에 액세스할 시각입니다. 12 단계 CNS의 배아 밴드 단축을 시작하고 단계 15 일까지 접합면의 확실한 패턴이 달성되었습니다. 단계 17으로 CNS 계약 및 PNS는 완전히 (캄포스 - 오르테가 외. 1985) 개발되고 있습니다. 따라서 PNS와 CNS의 패턴의 변화는 쉽게 이러한 발달 단계 동안 볼 수 있습니다.

Watch this Scientific Journal Video about Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos at JoVE.com

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

우리는 개최 준비하는 절차를 설명<em&gt; Drosophila</em&gt; embryogenesis 동안 배아 신경 시스템의 시각화를위한 배아.

전체 마운트의 배아 신경 계통의 시각화 Drosophila 태아

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

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Nanyang Technological University

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JoVE Journal

Neuroscience

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

13.2K 조회수.  SUNY-University at Buffalo. 우리는 개최 준비하는 절차를 설명 Drosophila embryogenesis 동안 배아 신경 시스템의 시각화를위한 배아.

비디오: Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

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.

Drosophila Melanogaster에서 애벌레 CNS의 해부

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

연구

생물학

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3 애벌레 Segmental 신경의 시각화 차 Instar Drosophila 애벌레 준비

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

신경과학

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

과일 플라이의 면역 염색과 Steroidogenic 기관 및 그들의 상호 작용하는 장기를 시각화하기위한 프로토콜<em&gt; 노랑 초파리</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

발생학

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

성인 표 피를 통해 초파리 다리 모터 신경 축 삭을 시각화

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

친유성 형광 염료를 사용하여 Drosophila 배아 모터 뉴런의 역행 추적

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

의 라이브 영상<em&gt; 초파리</em&gt; 애벌레 Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization(시신경엽 시각화를 위한 유충 및 성체 초파리 뇌의 해부, 면역조직화학 및 마운팅)

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

의 입식 이미징 Drosophila 태아

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

전체 마운트의 배아 신경 계통의 시각화 Drosophila 태아

요약

더 많은 비디오 탐색

이 비디오의 챕터

Drosophila Melanogaster에서 애벌레 CNS의 해부

3 애벌레 Segmental 신경의 시각화^차 Instar Drosophila 애벌레 준비

과일 플라이의 면역 염색과 Steroidogenic 기관 및 그들의 상호 작용하는 장기를 시각화하기위한 프로토콜

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

의 중추 신경계에 단일 세포의 라벨

성인 표 피를 통해 초파리 다리 모터 신경 축 삭을 시각화

친유성 형광 염료를 사용하여 Drosophila 배아 모터 뉴런의 역행 추적

의 라이브 영상

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization(시신경엽 시각화를 위한 유충 및 성체 초파리 뇌의 해부, 면역조직화학 및 마운팅)

의 입식 이미징 Drosophila 태아