이 작품은 R. 브루 스터 (1R01GM085290 - 01A1)에게 수여 NIH 교부금에 의해 지원되었다.

1.Microinjection <ol><li> 희석 플라스미드 인코딩 막 타겟 그린 형광 단백질 (mGFP, 리차드 할랜드의 호의) 40 NG / ML의 농도. DNA는 Qiagen의 맥시 준비 키트를 사용하여 정화 플라스미드 (리차드 할랜드의 mGFP/PCS2 +, 호의)를 linearizing으로 준비가되어 있습니다. 솔루션에 페놀 레드 (10분의 1 총 볼륨을 희석)의 추가는 솔루션을 시각화하는 것이 좋습니다. 얼음 DNA 솔루션을 유지. </li><li> 졸업 슬라이드에 바늘을 정렬하고 부드럽게 깨끗한 레이저를 사용하여 직경이 1μm는 바늘의 일부를 도청하여, stereomicroscope에서 주사 바늘을 (CAT 번호 NA - GD - 1 Narishinge 과학에서 90mm 유리 모세관) 보정 블레이드 (적절한 크기의 팁을 얻기 위해). </li><li> 알려진 볼륨 (2nl)을 삽입하기 위해 바늘을 보정하기 위해 microinjection 장치에 바늘을 연결 (PCI 100 Microinjector, 하버드 장치)와 앞에는 조각에 위치 페놀 레드와 물이 들어있는 솔루션 0.5μl으로 바늘을 기입 파라핀니다. 솔루션은 발을 페달을 사용하여 적절하게 사용하실 수 있습니다. 7 KPa로 압력을 설정하고 그것이 솔루션의 0.5μl 특면 250 페달 소요 있도록 (10msec 30msec) 시간을 달라집니다. </li><li> humidified 100mm 배양 접시에있는 왁스의 스트립에있는 눈금 바늘을 조심스럽게 넣습니다. 접시를 ...을 축이다하려면, 가장자리 주위에 서부 유럽 표준시 Kimwipes를 (이것은 바늘에있는 솔루션의 증발을 방지) 장소. </li><li> 바늘 끝 (보정하지 않은 끝)에 DNA 0.5 - 1μl를로드 micropipette을 사용합니다. </li><li> 이 솔루션은 바늘 끝에 도달되면, microinjection 장치에 로딩에 사용했던 바늘의 끝을 연결합니다. 발 페달을 사용하여 약 2 NL / 주사를 제공하기 위해 microinjector에 대한 설정을 조정합니다. </li><li> 엠브료 매체 (1.0 ML 행크의 주식 # 1, 0.1 ML 행크의 주식 # 2, 1.0 ML 행크의 주식 # 4, 95.9 ML ddH 2 O, 1.0 ML 행크의 주식 # 5 가득한 페트리 접시의 중앙에 다음 위치 zebrafish의 배아 , 1.0 ML 신선한 행크의 주식 # 6, 산도 7.2-10 방울 1 M NaOH에 대한 사용). </li><li> 태아가 원하는 단계로 개발한 후, 부드럽게 잘 포셉로 chorion를 손잡이 및 사출을위한 대상 지역에 바늘을 ...을 놓다. 넓은 범위 mGFP 표현은 1cell 단계에서 노른자 또는 세포질로 DNA를 주입. , 작은 지역에 mGFP 표현을 제한하는 하나의 세포의 세포질에 4 (이상) 세포 단계에서 DNA를 주입합니다. </li><li> microinjection 장치의 발을 페달을 누르면 약 2 NL을 제공, 솔루션 (페놀 레드의 추가로 인해 가시)가 제대로 분사는 것을 보장. </li><li> 조심스럽게 배아에서 바늘을 제거합니다. </li><li> 배양 접시에있는 모든 배아에 대해 반복합니다. </li><li> 배아는 28.5에서 원하는 단계 ° C를 개발하도록 허용합니다. </li><li> 부드럽게 잘 포셉을 (배아는 배아의 생존율을 증가 유리 페트리 접시에 dechorionating, 24hpf 미만 경우)를 사용하여 chorion를 벗겨내하여 Dechorionate 배아. </li><li> 유리 피펫을 사용하여 페트리 접시에서 빈 chorion를 제거합니다. </li></ol> 2. 이미징 mGFP - 표시 vibratome 섹션 <ol><li> 1X 인산 버퍼 용액 (PBS)에 정착액 (4 % paraformaldehyde로 배아의 수송을 유리 피펫을 사용합니다. 4 ° C 하룻밤에 배아를 수정. </li><li> 오분 각 1X PBS에서 배아 3 번 씻으십시오. </li><li> 페트리 접시, 그리고 좋은 집게를 사용하여 배아를 놓고, 배아를 손상시키지 않고 노른자를 제거합니다. 가능한 많은 노른자를 제거합니다. </li><li> H 2 O.에서 4% 낮은 용융 아가로 오스 준비 </li><li> 플라스틱 금형에 sectioning 플레이스 녹은 아가로 오스합니다. </li><li> 페트리 접시에서 주입된 배아를 리프트와 아가로 오스의 장소와 방향을 잘 집게를 사용하십시오. 배아는 주형의 한쪽 끝을 향해 배치되어야합니다. </li><li> 아가로 오스가 강화된되면, 금형의 아가로 오스 블록을 제거하는 등 주걱 같은 평평한 도구를 사용합니다. </li><li> 그것도되도록 면도날와 아가로 오스 블록의 아래쪽 끝부분을 잘라 버릴거야. </li><li> vibratome 무대 슈퍼 접착제 한 방울을 추가하고 그것에있는 아가로 오스 블록 (면도칼로 안정되었다 블록의 측면이 접착제에 직면한다)를 놓으십시오. 여러 개의 배아를 sectioning 들어, 무대에서 여러 아가로 오스 - 임베디드 표본을 탑재합니다. </li><li> (Vibratome 주식 회사) vibratome에 vibratome 무대를 연결합니다. </li><li> (:; 두께 - 50μm 속도 - 3 일반 설정) vibratome의 매개 변수를 설정합니다. 여러 개의 배아를 sectioning 경우, 무대 아가로 오스 블록을 정렬하고 (같은 배아에 해당하는 섹션이 함께 남아있게됩니다 것과 같은)으로 날개가 아가로 오스 블록의 전체 너비를 통해 전체 배아 통해 이뤄지는 아니지만되었는지 확인합니다. </li><li> vibratome 단계 1X PBS 하거라. </li><li> 섹션을 참조하십시오. </li><li> 일단 배아가 sectio입니다네드, vibratome 단계에서 아가로 오스 블록 (들)을 제거합니다. </li><li> 조심스럽게 아가로 오스 블록 (S)의 뒷면을 절단하여 각 섹션을 구분합니다. </li><li> 1X PBS - 채워진 멀티 잘 요리 (다른 배아에 해당하는 부분에 대해 서로 다른 우물을 사용)로 원하는 섹션과 장소를 선택하는 미세 집게를 사용하십시오. </li><li> 각각의 배아에 대해 반복합니다. </li><li> 항체 라벨을 진행, mGFP 영상 (섹션이 준비 하루 이틀 내에 몇 군데있다면이 단계가 필요하지 않을 수 있습니다) 향상시키기 위해, 23 불구하고 19 단계를 반복합니다. </li><li> 비 특정 바인딩을 차단하는 1X PBS에 차단 솔루션으로 실온에서 30 분 섹션을 처리합니다. </li><li> 기본 항체 (토끼 - GFP, Invitrogen 캣 번호 A11122) 4에서 5 상온에서 시간 또는 하루에 쉐이크에 차단 솔루션 @ 1:200 희석 ° C.와 함께 품어 섹션 </li><li> 1% BSA/1X PBS / 1퍼센트 DMSO 용액에서 네 번 (10 분, 15 분, 30 분 및 60 분 각) 씻으십시오. </li><li> 4 5 상온에서 시간 또는 숙박에 대해 차단 솔루션에 적절한 보조 항체와 섹션을 처리 ° C. </li><li> 1% BSA/1X PBS / 1퍼센트 DMSO 용액에서 네 번 (10 분, 15 분, 30 분 및 60 분 각) 씻으십시오. </li><li> 핵을 얼룩이 30 분에 대해, DAPI (Invitrogen 고양이 번호 D1306 50ml 1X PBS에 0.​​1 %의 DAPI)와 섹션을 처리합니다. </li><li> 오분 각 1X PBS로 세 번 씻으십시오. </li><li> 슬라이드에 마운트합니다. </li><li> 공촛점 현미경을 사용하여 이미지. </li></ol> 3. 라이브 영상 <ol><li> 그들은 원하는 발달 단계에 도달 Dechorionate 살고 주입 배아 삼십분 전에. 15 분 대한 진정 솔루션의 배아 (0.01 % 엠브료 매체 Tricaine)을 놓으십시오. </li><li> (; 부품 번호 P35G - O - 20 - C MatTek) 35mm 유리 바닥 문화 요리에 1% 아가로 오스의 드롭 놓습니다. </li><li> 아가로 오스는 응고 수 있습니다. </li><li> 아가로 오스 3 구멍 (예 : micropipette 팁 같은 다른 개체가 구멍을 만드는 데 사용할 수 있습니다)을 만들기 위해 화염을 통해 열 모세관. 여운 아가로 오스는 구멍 바닥에 없습니다 있는지 확인하십시오. </li><li> 엠브료 매체와 페트리 접시를 입력합니다. </li><li> 구멍에서 배아를 배치 유리 피펫을 사용합니다. </li><li> 다음 방향이 지역 (지느러미 머리) 몇 군데 수 있도록 좋은 집게를 사용하여 난자는 유리와 접촉하고 있습니다. </li><li> 조심스럽게 현미경 스테이지 (증발을 방지하기 위해 페트리 접시 위에 뚜껑을 장소)에 페트리 접시를 전송. </li><li> 이미징 동안 배아는 28.5 ° C.에 있는지 확인하십시오 </li></ol> 4. 검색 결과 우리는 여기서 모자이크 표현을 사용하여 zebrafish 신경 튜브의 이미지를 하나의 세포에 대한 직접적인 접근 방식을 설명합니다. zebrafish 배아의 광학 투명도에도 불구하고, 우리는 신경 세포의 시각화가 크게 vibratome를 사용하여 얻은 크로스 섹션에 향상된 것으로 나타났습니다. 이 섹션 공촛점 현미경을 사용하여 다른 초점 비행기에서 특정 세포에서 세포 이미징을위한 연장 허용 (50 ㎜) 두께입니다. 이 방법론을 사용하는 결과는 다른이 출판되어 있지만, WT 배아 (그림 1)과 N - cadherin (N - CAD)의 신경 튜브에 세포의 대표 이미지는 돌연변이 (그림 2)이 제공됩니다. 후자는 돌연변이 정중선 (그림 2)을 향해 제대로 방향에 실패 N - CAD에 신경 접시의 측면 지역에 세포를 보여줍니다. 이 모자이크 라벨과는 달리, 이러한 베타 catenin과 같은 일반적인 세​​포 표면 마커와 neuroepithelium을 immunostaining하면 각각의 세포의 형태는 (그림 3) 시각을 허용하지 않습니다. 라이브 배아의 시간 저속 영상은 neurulation 동안을 세포 역학의 이해를 가능하게 고정 표본의 모자이크 라벨을 보완. 영화 1 WT 세포가 적극적으로 medially 지향 막 라고나할까요을 확장하여 정중선으로 마이 그 레이션하는 것으로 나타났다. <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="그림 1" /> WT zebrafish 배아의 신경 튜브 그림 1. mGFP 표현. <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="그림 2" /> N - cadherin zebrafish 배아의 신경 튜브 그림 2. mGFP 표현. <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="그림 3" /> neuroepithelium 그림 3. B - catenin 단백질의 얼룩.

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Practical approach series. , (2002).</li><li>Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+is+required+for+the+polarized+cell+behaviors+that+drive+neurulation+in+the+zebrafish.">N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.</a> Development. 133, 3895-3905 (2006).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Motility+and+Actin+Cytoskeleton+of+Individual+Neurons+and+Neural+Crest+Cells+in+Zebrafish+Embryos.">Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos.</a> J Vis Exp. , (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

결론적으로, 분류 기술은 zebrafish 배아에 morphogenetic 프로세스의 단일 세포 분석을위한 수 있도록 여기에 설명되어 있습니다. 이 프로토콜의 주요 강조는 유비 쿼터스 모터의 제어하에 mGFP을 사용하여 신경 튜브의 영상 표시 전지 방식에 있습니다. 이 과도 표현 분석 독자의 추가 어플 리케이션을위한 안데르센 외의 최근 논문을 참조해야합니다. 3.

A correction was made to <a href="http://www.jove.com/index/Details.stp?ID=1976">Labeling and Imaging Cells in the Zebrafish Hindbrain</a>. There was an error in the authors affiliations. The authors have been corrected to:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster1 1Department of Biological Sciences, University of Maryland, Baltimore County 2Center for Neuroscience, Children's National Medical Center
instead of:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster2 1Center for Neuroscience, Children's National Medical Center 2Department of Biological Sciences, University of Maryland Baltimore County

A correction to the author affiliations has been made for the article Labeling and Imaging Cells in the Zebrafish Hindbrain.

Erratum: Labeling and Imaging Cells in the Zebrafish Hindbrain

솔루션 <ul><li> 행크의 주식 # 1 : 8.0 g NaCl, 100 ML DD H 2 O에서 0.4 g KCl </li><li> 행크의 주식 # 2 : 0.358 g 나 2 HPO 4 무수, 100 ML ddH 2 O에서 0.60 g KH 2 PO 4 </li><li> 행크의 주식 # 4 : 50 ML ddH 2 O에서 0.72 g CaCl 2 </li><li> 행크의 주식 # 5 : 50 ML DD H 2 O에서 1.23 g MgSO 4 x7H 2 O </li><li> 1 X 인산 버퍼 : 0.8 % NaCl, 0.02 %의 KCl, 0.02 M PO4, 산도 7.3 </li><li> 차단 솔루션 : 2퍼센트 정상 염소 혈청, 1 % 소 혈청 알부민 (BSA), 1 %의 dimethysulfoxide (DMSO) </li></ul>

labeling and imaging cells in the zebrafish hindbrain

초기 척추의 배아를 형성 morphogenetic 프로세스를 이해하는 데 핵심 고해상도로 이미지 세포 수있는 능력입니다. zebrafish 배아, 모자이크 표현의 플라스미드 DNA 결과의 주입이 단일 세포 또는 세포의 작은 클러스터의 시각화에 대한 허용에<sup&gt; 1</sup&gt;. 우리는 유비 쿼터스 모터의 제어하에 플라스미드 DNA 인코딩 막 타겟 그린 형광 단백질 (mGFP)의 분사가 neurulation를 겪고 이미징 세포에 사용할 수있는 방법을 설명합니다. 이 프로토콜에 센트럴 섹션에 있으며, 실시간으로 고해상도의 영상 표시 세포에 대한 방법론이다. 이 프로토콜은 젊은 zebrafish의 배아에 mGFP의 DNA의 삽입을 알아볼 수 있습니다. 배아는 다음 vibratome sectioning, 항체 라벨과 공촛점 현미경으로 영상에 대한 처리됩니다. 또는 mGFP을 표현 라이브 배아는 시간이 경과 공촛점 현미경을 사용하여 몇 군데하실 수 있습니다. 우리는 이전에 zebrafish 배아의 hindbrain 지역에서 신경 튜브 형성을 유발 세포 행동을 분석하는이 간단한 접근 방법을 사용했을<sup&gt; 2</sup&gt;. 라이브 영상은 neurulation 동안을 세포 역학의 이해를 가능하게이 방법을 보완하면서 고정 준비가 세포 모양과 신경 튜브 조직의 전례 시각화를위한있었습니다.

Watch this Scientific Journal Video about Labeling and Imaging Cells in the Zebrafish Hindbrain at JoVE.com

Labeling and Imaging Cells in the Zebrafish Hindbrain

초기 배아를 형성 morphogenetic 프로세스를 이해하는 데 핵심 고해상도로 이미지 세포 수있는 능력입니다. 우리는 여기서 세포막 타겟 그린 형광 단백질로 전체 zebrafish의 배아에서 세포의 단일 세포 또는 작은 클러스터를 라벨에 대한 기술을 설명합니다.

Zebrafish의 Hindbrain에 라벨 부착 및 이미징 셀

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

labeling-and-imaging-cells-in-the-zebrafish-hindbrain

Research

JoVE Journal

Neuroscience

11.2K 조회수.  University of Maryland, Baltimore County. 초기 배아를 형성 morphogenetic 프로세스를 이해하는 데 핵심 고해상도로 이미지 세포 수있는 능력입니다. 우리는 여기서 세포막 타겟 그린 형광 단백질로 전체 zebrafish의 배아에서 세포의 단일 세포 또는 작은 클러스터를 라벨에 대한 기술을 설명합니다.

비디오: Labeling and Imaging Cells in the Zebrafish Hindbrain

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

연구

신경과학

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

병아리 배아에서 축삭 궤적과 시냅스 대상을 추적하는 후뇌의 일렉트로

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

형광 염료와 성인 Zebrafish의 망막 신경절 세포의 퇴행성 라벨링

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

마우스 Hindbrain 배아 신생 공부에 대 한 모델

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

애벌레 Zebrafish의 측면 라인 세포의 생체 내 칼슘 이미징

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Sialoglycan의 신진 대사 표지에 의하여 1 차적인 신경 줄기 및 전구 세포의 세포 표면 마커를 확인

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

천연 폴리페놀 커큐민을 사용하여 뇌 조직에서 아밀로이드 플라크의 라벨링 및 이미징

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

두개골과 피부 제거에 의해 깨어 얼룩말 물고기 애벌레와 청소년에서 완전히 활성 뇌 조직의 생체 내 이미징

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...