This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

모든 동물의 절차는 미에 대학의 윤리위원회의 승인을하고, 국제 가이드 라인에 일본의 동물 복지 규제 &#39;복지와 동물의 관리에 관한 법률&#39;(일본의 환경부)에 따라 수행하고 준수 하였다. 니들 1. 준비 주 : 모든 실험은 마취 하에서 수행하고, 모든 노력이 고통을 최소화 하였다. 안락사, 생선 ≥20 분간 (≤4 ℃에서 5 부 얼음 / 물 1 부)에 빙수 욕에 침지 하였다. <ol><li> 바늘 풀러 (도 1a)과 1.0 mm의 외경 유리 모세관 당기는 유리 마이크로 모세관 바늘을 준비한다. </li><li> 비스듬하게 잘 가위 (그림 1B)를 사용하여 바늘의 끝을 잘라. 200 μm의 (그림 1C) - 이상적인 팁 직경은 약 100이어야합니다. 팁 diamete 경우R은 너무 좁고, 혈액 바늘을 입력하지 않습니다. </li><li> 5 ㎎ / ㎖의 농도로 염수에 헤파린을 녹인다. </li><li> 흡인기 튜브 어셈블리의 코 받침 이요 끝에서 미리 절단 바늘을 놓고 입에 마우스 피스를 보유, 또는 전구 지급기에 바늘을 연결합니다. 헤파린 용액에 바늘 끝을 담그고, 흡입에 의해 솔루션 (그림 1D와 1E)을 통해 불고 바늘을 heparinize. 주 : 흡인기 튜브 조립체는 제브라 피쉬 정자 동결 보존 (16)에 사용되어왔다. 긴 고무 튜브 입으로 비행하는 모든 혈액을 중지 할 수 있습니다. 관의 중간에 필터 설정 위험을 피할 수있다. </li><li> 적어도 1 시간 동안 10 cm 페트리 접시와 공기 건조에 헤파린 주사 바늘을 저장합니다. 바늘의 다수의 미리 제조 될 수있다. </li></ol> 2. 마취 <ol><li> (2- 페녹시 에탄올 100 ㎕ 물고기 200 ml의 물을 혼합하여 2 작은 플라스틱 케이스 마취 용액을 제조-PE). 마취제의 최종 농도는 500 ppm으로합니다. 주의! 2-PE는 빠른 효과가있다. </li><li> 순환 시스템에서 물고기 (AB 변형)의 원하는 번호를 제거합니다. </li><li> 2 분 (그림 1 층) - 1 마취제로 물고기를 전송하기 위해 인터넷을 사용합니다. 1 분 이내에 신속하게 아가미의 움직임을 점차, 수영, 수평 헐떡 거림을 가슴 지느러미를 확산하고,이 물고기를 관찰한다. </li><li> 시간이 지남에 따라, 물고기가 케이스의 바닥에 누워 관찰하고 마지막으로 수영을 중지합니다. 물고기가 말하다을 중지하고 아가미의 움직임이 느린 경우 마취의 수술 비행기에 도달합니다. 이 시점에서, 물고기는 혈액 수집을위한 준비가되어 있습니다. </li><li> 스키머가 부드럽게 2-PE에서 마취 물고기를 들어 올려 사용하면 마취제 (그림 1G)에 적신 종이 타월에 놓습니다. 눈의 건조를 방지하고 부드럽게하는 또 다른 부드러운 마른 티슈 페이퍼를 사용할 수있는 2-PE 용액으로 적신 부드러운 티슈와 물고기의 머리를 커버신체 표면을 건조. </li></ol> 3. 혈액 수집 <ol><li> 흡인기 튜브 어셈블리 (또는 전구 디스펜서)의 코 부분의 끝에서 헤파린 바늘을 놓고 입에 흡인기 튜브 어셈블리의 마우스 피스 끝을 잡고. </li><li> 코 부분의 끝과 함께 바늘을 잡고 조심스럽게 바늘 끝과 간섭 스케일을 제거합니다. 혈액 수집 사이트에 45 ° 각도 - 30 바늘을 삽입합니다. 위장관 (그림 1H)의 구멍을 피하십시오. 상술 한 바와 같이 전구 디스펜서를 사용하는 경우, 엄지 손가락과 가운데 손가락으로 전구를 눌러 먼저 손가락으로 전구의 끝 부분에 구멍을 차단 한 후 바늘을 삽입합니다. 참고 : 혈액 수집이 사이트는 지느러미 대동맥의 영역에서 항문에 몸 축과 후방을 따라입니다. 지느러미 대동맥 (DA) 및 후방 추기경 정맥 (PCV)은 척추에 복부 단지 (그림 2)입니다. </li><li> 빨아 시작바늘이 척추를 만지고 느낄 때 흡입 튜브 어셈블리의 끝을 마우스 피스. 피가 상승하지 않는 경우, 혈액의 흐름을 촉진하기 위해 손으로 미묘 바늘 끝을 이동합니다. 혈액이 바늘로 상승되면, 즉시 흔들어 중지하고 부드럽게 (그림 1I)을 빨아합니다. 전구 디스펜서를 사용하는 경우, 혈액을 흡인하는 전구의 압력을 해제. </li><li> 혈액 천천히 때문에 동맥 혈압 가능성 흡입없이 맥동 방식으로 바늘로 상승 할 것이다 관찰한다. 따라서 바늘이 제대로 동맥을 관통하면 흡인 할 필요가 없다. </li><li> 혈액의 적절한 볼륨이 수집 한 후 흡입을 중지합니다. 물고기에서 바늘을 제거하고 출혈을 멈추게하기 위해 부드러운 티슈를 사용하여 천자 사이트를 누릅니다. 손가락 압력의 20 초 (그림 1J) - 약 10 후 지혈을 준수하십시오. </li><li> 출혈이 정지 된 후, 즉시 CLE로 다시 전송 물고기따뜻한 물 (~ 28 ° C) 탱크. 이 수영을 시작할 때까지 부드럽게 아가미쪽으로 물을 소용돌이로 복구 할 수있는 물고기를 도와주세요. 참고 : 2 L 탱크에 5 postsampling 물고기까지 유지하고 물고기를 산소를하기 위해 순환 시스템에 탱크를 연결합니다. 물고기 물에 항생제를 첨가 할 필요가 없습니다. 일반 주택과 공급에 물고기를 유지한다. </li><li> 파라 필름의 조각 (그림 1K)의 깨끗한 영역에 바늘에서 피를 추방. </li><li> 상업적 휴대용 혈당 측정기 (도 1L)를 사용하여 혈당을 측정한다. 주 : 혈당 측정기는 글루코스 탈수소 효소 플라 빈 아데닌 디 뉴클레오티드 전극을 사용하고, 0.6 μL의 샘플 량을 필요로한다. <ol><li> 미터에 완전히 테스트 스트립을 삽입하고 직접 혈액 방울을 터치합니다. 자동으로 테스트 스트립에 혈액 무승부를 관찰하고 표시 영역에 혈당 결과 5 초를 얻을 수 있습니다. 결과를 기록하고 테스트 스트립을 폐기합니다. 사용각 측정을위한 새로운 테스트 스트립. </li></ol></li><li> (선택 단계) 피펫으로 혈액의 정확한 양을 가지고 더 분석 (헤모글로빈, 중성 지방 (TG), 총 콜레스테롤 등) microcentrifuge 관에 피를 전송합니다. 필요한 경우, 식염수 한 물고기에서 전체 혈액을 희석. RT에서 680 XG에서 3 분 동안 혈액 샘플을 원심 분리하여 플라즈마를 수확. 새로운 튜브에 플라즈마를 전송합니다. 지금부터, 생화학 적 분석에 사용될 준비가되어있다. </li></ol>

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The authors have nothing to disclose.

우리는 직렬 성인 제브라 피쉬에서 혈액을 얻기 위해 여기에 대한 자세한 프로토콜을 제시한다. 이 방법을 수행하는 것이 간단하며 우리는 일상적 실험에 사용. 이러한 혈액 수집 방법에 지브라 피쉬의 등쪽 대동맥 내로 유리 모세관 바늘 삽입에 근거한다. 이 절차를 수행하는 동안, 그것은 지느러미 대동맥를 검색하기위한 기준이 있기 때문에 척추를 브레이션 않도록주의하는 것이 중요합니다. ...

이들 장기 및 인간 유전학 1,2- 마찬가지이기 때문에 지브라 피쉬는 인간 질병의 유용한 모델로 증가 인기를 얻고있다. 발생 생물학의 분야에서 많은 연구는 제브라 피쉬와 인간의 쇼가 조혈 3, 지혈 4,5, 및 myelopoiesis 6 유사성을 표시 것을 증명하고있다. 성인 제브라 피쉬는이 모델 생물을 공유하기 때문에 인간의 질병에 혼란 그와 공통 경로를 면역 학적 7, 신경 퇴행성 8과 비만 관련 질병 (9)을 연구하는 데 사용됩니다. 비만 및 비만 관련 질환 (당뇨병, 간 지방증 및 비 알코올성 지방 간염 및 동맥 경화증), 제브라 피쉬 들어 혈당 및 지질 수준을 충분히 여러 형질 전환 및식이 유도 비만 모델 10-13에서 연구되었다. 개별 동물의 반복 채혈 동물 사용 및 12월을 줄일 수interindividual 차이를 rease. 그러나, 반복 된 시료 채취 때문에 상대적으로 작은 혈액량 쉽게 접근 용기의 부족 등 제브라 작은 동물에서 기술적으로 곤란하다. 이들 방법은 치사 포함 자신 단점을 가지고 있지만 제브라에서 일회성 채혈을위한 여러 방법이, 관련된 조직 손상 및 제한된 혈액량을 개발되어왔다. 예를 들어, 1-5 ㎕의 혈액 등쪽 대동맥 (5)의 영역의 길이는 약 0.3 cm의 횡 절개에서 수확 될 수있다. 팔 이음 뼈를 절단하여 가위로 잘린 5-10 μL 혈액 (10)를 수집 할 수 있습니다. 또 다른 편리한 혈액 샘플링 방법은 꼬리 어블 14이다. 심장 천자 동일한 물고기 반복 채혈을위한 하나의 잠재적 인 대안 방법이지만, 매우 소량이 절차 (약 50 NL)를 수득 할 수있다 perfor 분석의 수를 제한11 클럽 메드. 따라서, 새로운 프로토콜이 유기체는 인간의 질병을위한 표준 모델 생물하기 위해서는 중요한 미리 필요하다 반복적 치명적 혈액 샘플링을 가능하게하는데 필요하다. 이 기술은 약물 반응, 분자 진단용 바이오 마커, 예후 판정 발견과 같은 대사성 질환, 퇴행성 질환 및 악성 종양 등의 다양한 여러 종류의 질환의 모니터링을 테스트 할 수있다. 따라서 우리는 제브라 피쉬 직렬 15에서 혈액을 얻기위한 최소 침습적 방법을 개발했다. 여기에서 우리는 시각적으로 절차를 설명하고이 기​​술에 대한 자세한 프로토콜을 제공합니다. 이 방법을 사용하여, 다양한 파라미터를 포함 헤모글로빈, 공복 혈당 및 건강한 성인 지브라 피쉬의 혈중 지​​질에 기초하여 정상 값을 평가 하였다. 또한, 우리는 또한이 방법은 M에 의해 일련의 샘플을 필요로 연구에 적합한 지 여부를 평가과식 실험시의 혈당 변화를 시간적 onitoring.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass capillaries with filament</td>
			<td>Narishige</td>
			<td>GD-1</td>
			<td>1.0-mm-outer-diameter.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>To produce the needls</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>081-00136</td>
			<td>For heparinization</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Drummond</td>
			<td>2-040-000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>Bulb dispenser</td>
			<td>Drummond</td>
			<td>1-000-9000</td>
			<td>For blood collection</td>
		</tr>
		<tr>
			<td>2-phenoxyethanol</td>
			<td>Wako Pure Chemical Industries</td>
			<td>163-12075</td>
			<td>For anesthetizing the fish</td>
		</tr>
		<tr>
			<td>DRI-CHEM3500V</td>
			<td>Fujifilm</td>
			<td>-</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>DRI-CHEM Slides</td>
			<td>Fujifilm</td>
			<td>Hb-WII</td>
			<td>For hemoglobin measurement</td>
		</tr>
		<tr>
			<td>Glutest Neo Super</td>
			<td>Sanwa Kagaku Kenkyusho</td>
			<td>-</td>
			<td>For bood glucose measurement</td>
		</tr>
		<tr>
			<td>Wako L-type TG kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>464-44201</td>
			<td>For TG measurement</td>
		</tr>
		<tr>
			<td>Wako L-type CHO kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>460-44301</td>
			<td>For total cholesterol measurement</td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Alcan Packaging</td>
			<td>PM996</td>
			<td>To expel the blood on</td>
		</tr>
	</tbody>
</table>

repeated blood collection for blood tests in adult zebrafish

반복 채혈은 실험 동물에서 수행하는 가장 일반적인 방법 중 하나입니다. 그러나, 제브라 피쉬에서 채혈을위한 비 살상 프로토콜은 확립되어 있지 않다. 제브라 피쉬에서 혈액 수집에 대한 이전의 방법은 횡 절개, 잘린 꼬리 절제로, 치명적입니다. 따라서 우리는 여기에서이 절차를 요약 한 상세한 프로토콜을 소설 &quot;반복&quot;혈액 수집 방법을 개발하고, 존재했다. 이 방법은 침습적이고 따라서 동일인으로부터 반복 채혈이 가능 제브라위한 매우 낮은 사망률 (2.3 %)을 초래한다. 혈액 샘플링의 최대 볼륨은 물고기의 체중에 따라 달라집니다. 간격으로 반복 혈액 샘플링의 볼륨은 혈액의 헤모글로빈의 측정에 의해 평가 하였다 체중 매주 또는 ≤1 % 2 주마다의 ≤0.4 %이어야한다. 또한, 혈색소, 공복 혈당, 혈장 중성 지방 (TG), 총 C남성 및 여성 성인 holesterol 지브라 피쉬의 수준을 측정 하였다. 우리는 또한 다이어트 유발 된 비만에서 포도당 대사의 조절 곤란을 조사하기 위해이 방법을 적용했다. 이러한 혈액 수집 방법은 글루코스 및 지질 대사 및 인간의 질환 모델 생물로서 지브라 피쉬의 사용을 증가시킬 것이다 혈액 학적 연구를 포함하는 많은 애플리케이션을 허용 할 것이다.

(도 1J를 &lt;1mm 천자) 및 2.3 %의 매우 낮은 치사율을 산출 이러한 혈액 수집 방법은 제브라 최소한 부상시킨다. 우리는 하나의 물고기에서 수집 및 체중 (그림 3)의 관계를 평가 될 수있는 혈액의 최대 볼륨을 조사했다. 우리는 수집 된 최대 혈액량이 선형 적으로 체중 (R = 0.813) 상관 관계를 보였다 것으로 나타났습니다. 개별 물고기 (체중 = 1.071 g)으로부터 혈액 채취 큰 부피...

Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

성인 Zebrafish의에서 혈액 검사에 대한 반복 채혈

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.5K 조회수.  Mie University. Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

비디오: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

미국 호르세쇼 게, Limulus Polyphemus의 혈액 수집

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

연구

생물학

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

생성중 성인 Zebrafish 테일 핀으로 Morpholinos의 생체내 Electroporation에

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

성인 Zebrafish의를 Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

키나제 억제제에 대해 내성 돌연변이의 검사 및 검증을위한 방법

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

유동 세포 계측법을 사용하여 세포 외 소포의 분석을위한 기술

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).