이 작품은 NIAID에서 NRM 및 JLB에 SREB의 논문의 화목 상을 부여하는 번호 R01AI055001 및 R01AI082127 지원했다.

1. 플루오레신는 dextran과 GSL I - isolectin B 네 얼굴 정맥 주사가 배아 vasculature를 라벨에 표시 <ol><li> isolectin B 1.5mL Eppendorf 튜브에 PBS에서 4 (20ug/200uL) 37에 따뜻한 ° C. - 인산에 FITC - dextran (50ug/mL)를 준비 식염수 (PBS) 또는 GSL 1 버퍼 그린 / PBS GSL 1 FITC - dextran 솔루션 (총 볼륨 1mL) 및 주식 1.25mM 빨리 180uL 주식 1.25mM 빠른 그린 / PBS의 100uL 추가 - isolectin 솔루션은되도록 B 4 (총 볼륨 200uL) 눈에 파란색. </li><li> allantoic 스토킹 (제대 동맥과 정맥)은 그대로두고, 함께 E14.5 - E18.5 배아와 난황을 해부하다. </li><li> 새로운 petrie 요리 (60 X 15mm)로 배아를 전송하고 실온에서 PBS에 젖어. </li><li> 머리 / 얼굴의 화살보기를 제공하기 위해 배아를 놓습니다. 부드럽게 머리에있는 배아를 파악하기 위해 마이크로 해부 집게를 사용하십시오. </li><li> 30G 바늘을 사용 50uL FITC - dextran (50ug/mL) 또는 GSL 1 삽입 - isolectin B 4 (200uL PBS에서 20ug)를 머리 뒤쪽 방향으로 바늘을 가리키는 얼굴 정맥에. </li><li> 염료가 제대 정맥에서 볼 때, 바늘을 제거하고 all​​antoic 스토킹 (제대 동맥과 정맥)에서 배아를 구분한다. </li><li> 주사 다음, 배아는 염료가 배아를 통해 순환되도록 2~3분에 대한 실온에서 PBS에 남아있을 수 있습니다. </li></ol> 2. 피부 vasculature 전체 마운트 분석 <ol><li> 염료는 배아에 걸쳐 순환 수 있도록 후, 다시, 사지의 지역에서 피부 샘플을 제거하고 복부 등 8,9. </li><li> 차가운 PBS의 피부 샘플을 씻고, 그리고 이시간 8,9에 대한 4% PFA / PBS에 수정. 십분 2mL 차가운 PBS로 4mL 명확 약병에 각각에 대해 3 번 씻으십시오. </li><li> 현미경에 놓습니다 스킨 샘플 100 각 슬라이드에 미디어를 장착의 μl와 커버 유리 슬라이드 및 추가합니다. </li><li> 슬라이드는 어두운 저장 영역에서 건조하도록 허용합니다. </li><li> 2 &#39;이미지 수집&#39;섹션 단계로 진행합니다. </li></ol> 3. thymus와 심장 vasculature 및 cryosections에 대한 perivascular 세포의 멀티 컬러 라벨은 (제 1, 7 단계에서 계속) <ol><li> 액체 질소에서 &#39;플래시 프리즈&#39;전체 배아. 공룡의 태아가되며 -80 ° C에서 분석까지 저장할 수 있습니다. </li><li> 또는, thymus을 해부하다 4 ° C PBS로 씻어, 2 시간 동안 2mL 4 % Paraformaldehyde (PFA) / PBS에 수정. -80에서 사용 ° C. 때까지 차가운 PBS, 10에서 개최 thymi하고, 동결 및 상점에서 10 분 동안 3 번 씻으십시오 </li><li> cryosectioning 들어, 섹션에서 &#39;블록&#39;에 OCT를 확산하고 sectioning에 대한 배아 또는 해부 기관 / 티슈를 탑재합니다. </li><li> 10 μm의 두께는 섹션으로 냉동 조직을 잘라 슬라이드에 수집합니다. </li><li> 50~10분위한 아세톤의 섹션을 수정. 추운 TBS에서 3 번 씻으십시오. </li><li> 실온에서 습도 챔버에서 10 % 당나귀 혈청 / TBS에 차단합니다. </li><li> 4 습도 챔버의 주요 항체 100 μl 1 1 시간 하루에 대한 섹션을 품어 ° C : 예제에서, 우리는 쥐의 반 마우스 CD31 (1:100) 라벨 내피하고, 염소 반 마우스 PDGFR - β를 사용하여 (1:100) perivascular 세포를 분류합니다. 이것은 항체가 균일 섹션에 걸쳐 확산되도록하기 위해 개별적으로 절단 Parafilm 스트립과 함께 슬라이드를 커버하는 데 유용합니다. </li><li> 주요 항체와 부화 다음, 차가운 TBS에서 섹션에게 3 회 씻는다. 최소 30 분 적절한 보조 항체의 100 μl로 품어. </li><li> 추운 TBS에서 3 번 씻으십시오. 100 각 슬라이드에 미디어를 장착의 μl와 덮개 유리를 추가합니다. </li><li> 슬라이드는 어두운 저장 영역에서 건조하도록 허용합니다. </li><li> &#39;이미지 수집&#39;섹션을 참조하십시오. </li></ol> 4. thymus vasculature 및 vibratome 섹션에 대한 perivascular 세포의 멀티 컬러 라벨은 (제 1, 7 단계에서 계속) <ol><li> 배아로부터 thymus 엽 (叶)을 해부하다 차가운 PBS로 린스. </li><li> 2 시간 동안 실온에서 4% PFA / PBS에 thymus을 수정. </li><li> 용융 낮은 4%의 작은 플라스틱 카트리지와 잠수함의 PBS - 트리톤 X (0.15 %) 3 시간, 10 분 장소 thymi에 씻으 아가로 오스 / PBS (~ 4 ° C). thymus은 카트리지의 하단에 접촉해야합니다. </li><li> 아가로 오스는 얼음 (3~5분)에 응고 수 있습니다. 초과 아가로 오스를 잘라하기 위해 면도날을 사용합니다. vibratome 블록에 접착제를 추가하고 차단하는 샘플을 준수합니다. </li><li> 샘플 및 블레이드가 포장되어 때까지 vibratome 물을 욕조에 차가운 PBS를 추가합니다. </li><li> 속도와 진폭을 (높은 진폭과 낮은 중간 속도 소프트 thymus 섹션에 이상적입니다) 설정합니다. 섹션 초과 교반으로 인해 헤어질 경우 진폭은 감소한다. </li><li> 50 음 섹션을 잘라 버릴거야. </li><li> 붓을를 사용 차가운 PBS에 24 잘 microplate에 섹션을 수집합니다. </li><li> 30 분 동안 PBS - 트리톤 X에서 10 % 당나귀 혈청 (0.15 %) 500 μl의 블록 섹션. </li><li> 같은 차 항체와 함께 하루를 8 시간에 대해 품어 섹션 안티 CD31 및 안티 - PDGFR - β, 대상 24 잘 microplate 인치 </li><li> 4 8 시간 총 ° C.여 PBS - 트리톤 X 3 회 (0.15 %)를 씻으십시오 </li><li> 30 분 동안 PBS - 트리톤 X에서 10 % 당나귀 혈청 (0.15 %)에서 블록 섹션. </li><li> 4 ° C와 함께 적절한 보조 항체에 하루에 8 시간 섹션을 품어. </li><li> 4 8 시간 총 ° C.여 PBS - 트리톤 X 3 회 (0.15 %)를 씻으십시오 </li><li> 다시 수정 샘플 4%에 PFA / 얼음에 30 분 PBS. </li><li> 얼음 30 분 이상의 PBS - 트리톤 X 3 회 (0.15 %)를 씻으십시오. </li><li> 25% MeOH, 50 % MeOH, 75 % MeOH, 각 단계를위한 10 분 100 % MeOH : 등급 MeOH / PBS - 트리톤 X 시리즈를 통해 샘플을 탈수. 십분 후에 신선한 MeOH로 100 % MeOH를 교체하고 실온에서 1 시간 품어. </li><li> 유리 용기에서 혼합 BABB (벤질 알코올 : 벤질 벤조 산) 1시 2분 비율 인치 50 % BABB와 50 % MeOH의 최종 농도에 대한 MeOH와 BABB을 결합합니다. 10-15분에 대한 MeOH : BABB에서 샘플을 품어. </li><li> 상온에서, 10-15 분 또는 정리 할때까지 백퍼센트 BABB과 부화로 유리 용기 샘플을 전송합니다. </li><li> 측면으로 신선한 100 % BABB 및 전송 샘플과 우울증 슬라이드 (0.7mm 깊이)를 입력합니다. 매니큐어로부터 2-3 코트와 유리 (제 1.5) 및 인감을 커버 추가합니다. 상온에서 어둠의 강화 매니큐어가 손톱 허용 후 4 샘플을 저장 ° C. 참고 : 슬라이드가 완전히 공촛점 이미지 수집하기 전에 봉인해야합니다. 형광 염료가 BABB에 페이드 수있는 이미지는 12~24시간 이내에 취득해야합니다. </li><li> &#39;이미지 수집&#39;섹션을 참조하십시오. </li></ol> 5. 이미지 수집 <ol><li> 이미지 10 488과 계획 - 고차 색지움 20X/0.8 목표를 (512 X 512 픽셀)를 사용 공촛점 현미경으로 μm의 냉동 섹션 - (FITC-dextran/GSL 1 - B 사 isolectin), 543 -, 그리고 633 - NM 레이저 라인. </li><li> 공촛점 취득 전체 마운트 피부 및 50 μm의 488과 계획 - 고차 색지움 10X/0.4 목표를 (512 X 512 픽셀)를 사용하여 아가로 오스 - 임베디드 섹션의 Z - 섹션 - (FITC-dextran/GSL 1 - B 사 isolectin), 543 - 와 633 - nm의 레이저 라인. 시리얼 Z - 부분은 각 각 채널에 대해 1 미크론에서 순차적으로 수령해야합니다. </li><li> 자이스 혈구 Axiovision 4.6이나 기타 이미지 분석 소프트웨어를 사용하여 Z - 섹션을 직렬 재구성. </li></ol> 6. 대표 결과 : 배아 vasculature의 효율적인 라벨링은 배아 생쥐에서 혈관 결함을 평가하기위한 중요합니다. 그림 1은 각각 좌우 심실의 얼룩 (1E - F) 이외에 E16.5 thymus 혈관 (1A - B)와 CD31 (1B)와 함께 공동 상표, 특정 라벨을 보여줍니다. GSL I - isolectin B와 같은 섹션 1, 3, 5에 설명된 cryosections 4 프로토콜은 이러한 실험에 사용되었다. 전체 마운트 E16.5 생쥐의 피부 혈관 네트워크의 레이블, 섹션 1, 2에서 설명된 프로토콜을 사용하여, 5은 그림 1C - D에 표시됩니다. <img alt="그림 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> 한 전설을 그림. FITC GSL I - E16.5 마우스 배아에 isolectin B 네 얼굴 정맥 주사. 배아 피부 vasculature 다음 주입의 주입 다음 배아 thymus의 A.의 Cryosection. isolectin B 4 CD31 공동 상표의 B. 병합합니다. C.와 D. 전체 마운트. E. 및 배아 심장의 E. F.의 Cryosection (오른쪽 뇌실) F. (뇌실 왼쪽) 다음과 같은 분사.

<ol>
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관심 없음 충돌 선언하지 않습니다.

섹션에서 전체 마운트 및 PECAM - 1 (CD31) 얼룩은 배아 생쥐에서 vasculature을 라벨에 대한 종래의 방법입니다. 이 방법은 직접 및 / 또는 간접 immunofluorescence와 마우스 조직을 permeabilize하기 위해 세제의 사용을 필요로합니다. 이것은 다소 적시에 처리하는 증명한다. 여기, 우리는 FITC - dextran 또는 isolectin B 사 안면 정맥 주사 직접함으로써 항체 라벨 단계에 대한 요구를 제거, 배아 vasculature 레이?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> FITC - dextran </td><td> 시그마 </td><td> FD150S - 1G </td></tr><tr><td> isolectin B 4 - 플루오레신는 GSL 1 분류 </td><td> 벡터 연구소 </td><td> FL - 1201 </td></tr><tr><td> 빠른 그린 </td><td> MP의 Biomedicals </td><td> 195,178 </td></tr><tr><td> PFA </td><td> Fluka </td><td> 76,240 </td></tr><tr><td> 태아 소 혈청 </td><td> 애틀랜타 체액 </td><td> S11550 </td></tr><tr><td> 최적의 절삭 온도 컴파 운드 (OCT </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> 아세톤 </td><td> JT 베이커 </td><td> 9006-33 </td></tr><tr><td> 당나귀 세럼 </td><td> 잭슨 </td><td> 017-000-121 </td></tr><tr><td> 쥐 방지 마우스 CD31, </td><td> BD Pharmingen </td><td> 558,736 </td></tr><tr><td> 염소 안티 마우스 PDGFR - β </td><td> R &amp; D 시스템 </td><td> AF1042 </td></tr><tr><td> 당나귀 안티 쥐 CD31 알렉사 647 (Invitrogen) </td><td> Biolegend </td><td> 102,516 </td></tr><tr><td> 당나귀 안티 염소 알렉사 594 (Invitrogen) </td><td> Invitrogen </td><td> A11058 </td></tr><tr><td> 트리톤 X -100 </td><td> 시그마 - 올드 리치 </td><td> X - 100 </td></tr><tr><td> 낮은 용융 아가로 오스 / PBS </td><td> 시그마 - 올드 리치 </td><td> A9414 - 25G </td></tr><tr><td> 메탄올 </td><td> 피셔 과학 </td><td> A413 - 4 </td></tr><tr><td> 벤질 알코올 </td><td> Acros 과학 </td><td> 148,390,010 </td></tr><tr><td> 벤질 벤조 산 </td><td> Acros 과학 </td><td> 105,860,010 </td></tr><tr><td> 우울증 슬라이드 </td><td> 피셔 과학 </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> 전자 현미경 과학 </td><td> 17985-10 </td></tr><tr><td> 커버 유리 (22X22) -1.5 </td><td> 써모 과학 </td><td> 152,222 </td></tr><tr><td> 자이스 혈구 LSM 510 메타 공촛점 현미경 </td><td> 자이스 혈구 </td><td></td></tr><tr><td> 마이크로 해부 포셉 </td><td> Roboz </td><td> RS - 5135 </td></tr><tr><td> Parafilm 번호 OM992 </td><td> 피셔 과학 </td><td> 13-374-16 </td></tr><tr><td> 12 24 잘 microplates </td><td> 에버그린 과학 </td><td> 222-8044 - 01F </td></tr><tr><td> Superfrost / 플러스 현미경 슬라이드 </td><td> 피셔 과학 </td><td> 12-550-15 </td></tr><tr><td> 4mL 맑은 튜브 </td><td> 국립 과학 </td><td> B7800 - 2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

기능성 혈관 네트워크의 설립 organogenesis의 필수적인 부분이며, 최적의 장기의 기능이 필요합니다. 예를 들어, thymus 적절한 vasculature 형성과 patterning에있는 기관 및 주변에 성숙한 T 세포 출구로 thymocyte 입학 필수적입니다. thymus에서 혈관의 공간적 배열은 로컬 microenvironment의 신호, 즉 상피 세포 thymic (TEC)에 의존적일 수 밖에 없습니다. 몇 가지 최근의 보고서는 thymus 혈관 결함 1,2에서 이러한 신호 결과의 중단을 제안한다. 이전 연구는 신생아와 성인의 thymus vasculature 1,2 레이블을하는 데 사용되는 기술을 설명합니다. 우리는 여기서 배아 thymus에서 혈관을 라벨에 대한 기술을 보여줍니다. 이 방법은 FITC - dextran 또는 Griffonia (Bandeiraea) Simplicifolia 렉틴 I (GSL 1 - isolectin B 4)의 사용을 결합한 얼굴 정맥 주사 및 CD31 항체 얼룩 thymus 혈관 구조와 thymic perivascular mesenchyme 3-5 라벨 PDGFR - β를 식별할 수 있습니다. cryosections 또는 vibratome 섹션을 사용할 수있는 옵션도 제공됩니다. 이 프로토콜은 thymus 혈관 형성에 TEC - 파생 분자의 역할을 정의하는 것이 중요합니다 thymus 혈관 결함을 식별하는 데 사용할 수 있습니다. 방법은 전체 vasculature를 레이블로, 그것은 또한 피부와 마음 60-10 포함한 배아에 걸쳐 여러 기관과 조직에 혈관 네트워크를 분석하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

이 문서는 배아 피부와 thymus 혈관을 라벨에 대한 방법을 설명합니다.

배아 마우스에 Vasculature을 라벨링하는 방법

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

a-method-for-labeling-vasculature-in-embryonic-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.5K 조회수.  University of Georgia. 이 문서는 배아 피부와 thymus 혈관을 라벨에 대한 방법을 설명합니다.

비디오: A Method for Labeling Vasculature in Embryonic Mice

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

해제 배아 마음에 관상 동맥 혈관의 분석

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

연구

발생학

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

의 배아 혈관 신생에 대한 시각화 및 정량 분석<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

혈관을 조사하기 위해 신생아 마우스 망막의 전체 마운트 면역 형광 염색<em&gt; 생체 내</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

신경과학

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

전체 산 Epifluorescence를 사용 하 여 마우스 Embryogenesis 동안 심장 챔버의 분석

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

생쥐 배아에서 초기 심장 전구체의 생생한 영상

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

배아 림브 스킨 Vasculature에 대한 전체 마운트 Immunohistochemical 분석 : 엠브료에 혈관 분기 Morphogenesis를 공부하는 모델 시스템

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

생물학

Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one of the first organs to start functioning during development. Mechanisms of blood vessel formation have become a subject of high scientific and clinical interest. In adults however, it is difficult to visualize the vasculature in most living animals due to their localization deep within other tissues. Nevertheless, visualization of blood vessels remains important for several studies such as endocrinology and neurobiology. While several transgenic lines have been developed in zebrafish, with blood vessels directly visualized through expression of fluorescent proteins, no such tools exist for other teleost species. Using medaka (Oryzias latipes) as a model, the current protocol presents a quick and direct technique to label blood vessels in brain and pituitary by perfusing through the heart with fixative containing DiI. This protocol allows improvement of our understanding on how brain and pituitary cells interact with blood vasculature in whole tissue or thick tissue slices.

The article describes a quick protocol for labeling blood vessels in a teleost fish by cardiac perfusion of DiI diluted in fixative, using medaka (Oryzias latipes) as a model and focusing on brain and pituitary tissue.

텔 레 Ost 뇌와 뇌 하 수 체에 있는 혈관의 라벨링은 DiI와 함께 심장 관류를 사용 하 여

Blood vessels innervate all tissues in vertebrates, enabling their survival by providing the necessary nutrients, oxygen, and hormonal signals. It is one ...

생체공학

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

마우스의 심장 형태학의 echocardiographic 및 조직학 검사

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

의학

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

배아의 마우스 하트을 개발에있는 셀의 레테르를 붙이는 및 사출

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

배아 마우스에 Vasculature을 라벨링하는 방법

요약

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