The authors have nothing to disclose.

모든 절차는 &quot;실험 동물 관리의 원칙&quot;에 설명 된 지침에 따라 수행된다 (건강 간행물의 국립 연구소 없음. 85-23, 1985 년 개정) 및 승인 로잘린 프랭클린 대학 기관 동물 관리 및 사용위원회의 동물 프로토콜은 08-19. 머리 세포 괴사의 1. 겐타 마이신 유도 <ol><li> 0.004 %의 최종 농도 (4.32 ㎜)에 생리 식염수에 황산 젠타 마이신을 준비한다. </li><li> 0.004 % (4.32 ㎜) 겐타 마이신 용액이 들어있는 용기에 어른 물고기 (D. rerio, 연령 4 ~ 6 개월) 놓습니다. 모든 컨테이너를 사용할 수 있습니다,하지만 우리는 폭 7, 고 6, 긴 7 인 Pharmacal 물 시스템에서 물고기 컨테이너를 사용합니다. 24 시간 동안 28 ° C에서 설정 인큐베이터에서 물고기와 컨테이너를 배치합니다. 24 시간 동안 실행 가능한 상태로 물고기를 유지하기에 충분한 수준으로 탱크 내의 유체의 총 체적을 설정한다. 참고 : 겐타 마이신 유체의 통기가 nece하지 않습니다ssary 충분한 볼륨이 치료되는 어류의 번호를 사용하는 경우. </li></ol> 머리 세포의 2. 생명 염색 <ol><li> 15 ㎎ / ㎖ 작업 스톡 용액으로부터 형광 중요한 염료 [4-4 - 디 에틸 아미노)-N-메틸 피리 디늄 요오다 이드 (485 nm의 여기 λ 및 메탄올 603 nm의 발광 λ)의 (정상 식염수) 0.08 % 농도를 준비 에탄올. </li><li> 젠타 마이신 치료가 치료 물고기가 중요한 염료를 포함하는 6 웰 배양 플레이트의 우물에서 물고기를 배치하여 즉시 얼룩 져 제어 및 겐타 마이신의 일부 효과가 있는지 확인하십시오. 그 물고기는 75 분 동안 염색되지 않도록 통계적 유의성이 달성 될 때까지 필요에 따라 충분한 물고기의 수 (및 배양 플레이트를 사용합니다. neuromast 계산의 심사관의 속도에 따라, 시간이 지남에 따라 지그재그 방식으로 접시에 물고기를 배치 단계 2.3에 기재된대로. </li><li> 형광 현미경에 의해 벤치 서랍 단계 2.2에서 접시를 놓습니다스테인드 neuromasts의 검사에 사용. 실온에서 1 시간 염색에 걸쳐 중요한 염료의 냉각을 방지하기 위해 실내 조명을 끄십시오. </li><li> 염료 세척 아웃 마취 물 탱크를 모두 준비합니다. 염료 씻어 ​​아웃 물은 일반 물 물고기이며, 보통 물고기 물에 1:1,000 희석이 달성되도록 마취 물, 충분한 2 - 페녹시 에탄올을 추가합니다. </li><li> 초과 중요한 염료를 씻어 중요한 염료 염색 물고기의 관찰 3.1 단계로 진행하기 위해 여분의 일반적인 물고기 물에서 물고기를 놓습니다. </li><li> 28 ℃에서 8-16 사이의 시간 동안 인큐베이터에 일반 물고기 물에 세척 겐타 마이신 처리 된 생선을 전송, neuromasts의 재생을 검사하려면 </li><li> 8-16 시간 사이에 여러 번, 물고기, 인큐베이터에서 제거 세척 단계 2.1-2.4에 표시된 스테인드됩니다. 중요한 염료 염색 물고기의 관찰 3.1 단계로 진행합니다. </li></ol> 3. 마취 생선 Neuromasts의 형광 계수 <ol><li&gt; 과량의 유체를 제거하고 플라스틱 페트리 접시 뚜껑의 중앙에 여과지 적신 장에 배치하는 종이 타월에 각각 물고기 럽히. </li><li> 중앙 신체 바늘의 중요한 염료 염색 neuromasts의 디지털 이미지를 얻기 위해 형광 실체 현미경의 무대에 뚜껑을 놓습니다. </li><li> 사용하는 형광 실체 현미경에 배치 된 디지털 카메라는 이후의 정량 분석​​을위한 이미지를 캡처하는 배의 배율을 설정합니다. 실체 현미경의 배율 설정이 사용되는 현미경의 브랜드에 따라 달라질 수 있지만, 설정은 중앙 신체 바늘에서 쉽게 볼 수 있도록 개별 neuromasts의 계산을 허용해야합니다. </li><li> (그림 1 참조) 오른쪽 가슴 지느러미에 바로 인접 물고기의 가장 아래 복부 측면에있는 4 개의 지정 바늘에서 볼 수 neuromasts의 수를 계산하여 재생의 양을 결정합니다. 통계 분석을 위해 같은 ANOVA 오 같은 적절한 테스트를 사용연구 학생의 T-테스트. 실험 시점 당 5 물고기의 최소값을 활용해야하고 모든 실험은 3 배의 최소 반복되어야한다. </li><li> neuromast 재생 시간 곡선을 바탕으로 (그림 3 참조), 재생 곡선의 선형 위상 내에 8-16 시간 포스트 겐타 마이신 씻어 아웃 사이 neuromasts을 계산합니다. 참고 : 선형 시간 단계의 사용은 제어 및 실험 그룹간에 적절한 정량 분석​​을 할 수 있습니다. </li></ol> Neuromast 분석은 통계적으로 유의하지 않은 경우 정량 분석​​의 높은 해상도를 얻기 위해 개별 모발 세포의 4. 형광 계수 <ol><li> neuromasts의 레벨에서 정량적 분석이 중요하지 않은 경우에, 개별 모발 세포 수준에서의 분석은 또한 해상도가 높은 수준을 얻기 위해 이용 될 수있다. 특정 시점 포스트 겐타 마이신 씻어 아웃 (시점을 이전 neuromast 연구 기준), 생명 색소 성에서 물고기를 선택이 프로토콜에 설명 된 물고기를 아인 후 1-5 분 동안 1:500 희석액에 2 - 페녹시 에탄올을 사용하여 물고기를 안락사. </li><li> 다음과 같이 사각형 피변 준비가 이루어 지도록 담금질 않도록 부드러운 조명에서, 네 개의 절개를 만든다. 이 뒷 지느러미와 정렬 될 때까지 물고기의 상부 늑골을 따라 절개를 한 후 배에서 절개를하고, 마지막으로, 그래서 광장 피부 플랩이 만들어 이러한 절개의 양쪽에있는 두 개의 수직 절개를합니다. 참고 :이 피부 준비 neuromast 실험에 사용 된 중간 바디 스티치를 통합 할 것입니다. </li><li> 유리 슬라이드에 피부 표본을 놓고 다음 앵커를하는 데 도움이 이후의 디지털 이미징을위한 조직을 평평하게 절제 피부 표본을 통해 원형 유리 커버 슬립을 배치합니다. </li><li> 단계 8.6에서 피부 표본을 사용하여, 중간 품 스티치 각각 neuromast 내의 유모 세포의 디지털 이미지를 얻는다. 적어도 60 배의 배율로 이미지를 가져온 후 머리 CE를 계산제어 및 실험 그룹의 비교 정량 분석에 대한 개별 neuromasts 내 LLS (그림 4 참조). </li></ol>

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D., Louise Glass, ., L, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-Cell+Imaging+of+Vegetative+Hyphal+Fusion+in+Neurospora+Crassa.">Live-Cell Imaging of Vegetative Hyphal Fusion in Neurospora Crassa.</a> Fungal. Genet. Biol. 37 (1), 109-119 (2002).</li><li>Olsen, A. S., Sarras, M. P., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+Regeneration+Is+Impaired+in+an+Adult+Zebrafish+Model+of+Diabetes+Mellitus.">Limb Regeneration Is Impaired in an Adult Zebrafish Model of Diabetes Mellitus.</a> Wound Repair Regen. 18 (5), 532-542 (2010).</li><li>Olsen, A. S., Sarras, M. P., Leontovich, A., Intine, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+Transmission+of+Diabetic+Metabolic+Memory+in+Zebrafish+Correlates+With+DNA+Hypomethylation+and+Aberrant+Gene+Expression.">Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression.</a> Diabetes. 61 (2), 485-491 (2012).</li></ol>

This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

배아와 애벌레 zebrafish의 8,24,25의 옆 선 (LL) 중생의 분석을 위해 설립 된 문학의 광대 한 몸을 바탕으로, 우리의 연구의 목표는 제브라 피쉬의 옆 선 재생을위한 정량적 분석을 개발하는 것이라고 할 수 최고의 성인 물고기에서 공부하는 질병 모델에 적용 할 수. 우리는 성인 물고기 배아 / 애벌레 물고기를 위해 개발 절차를 적용 할 때 특정 중요한 포인트가 중요하다는 것을 발견했다. 이?...

옆 선 (LL) 시스템은 청각, 균형, rheotaxis하고 학교와 포식자 회피 1-5으로 중재 행동에 대한 책임이 모두 물고기와 양서류에서 발견 mechanosensory 기관이다. 이는지지 세포에 의해 둘러싸여 유모 세포의 클러스터로 구성되며, 둘 모두는 neuromasts 6 불리는 구조에 배치된다. 이 neuromasts는 일반적으로 물고기의 머리에서 관찰 된 일부 수평 바늘 몸과 꼬리의 길이 방향 축을 따라 (바늘라고 함)의 수직 라인으로 구성되어 있습니다. 성인에서 neuromasts는 배아 또는 애벌레 물고기 6에 비해 바늘 내 번호 상당히 크다. 제브라 피쉬의 생물 의학 연구는 항생제 치료, 소음에 의한 외상, 만성 감염 등의 효과에 초점을 맞추고있다. 유모 세포에 시도에서 7,8 더 나은 인간에 미치는 영향을 이해합니다. 대부분의 척추 동물과는 달리, 테이러한 제브라 피쉬 (다니오 rerio) 등 leosts는 손실 유모 세포를 재생하는 기능이 있습니다. 제브라 피쉬 때문에 자신의 빠른 개발 시간과 높은 재생 능력으로 특히 유용하다. 그러나 현재; 옆 선 개발 및 / 또는 재생에 제브라 피쉬의 연구는 주로 쉽게 계산 및 분석 6,9,10 수 있습니다 옆 선 neuromasts의 감소 번호로 배아 및 애벌레 단계의 물고기를 활용했다. 그러나, 신경과 비 신경 질환 11-16의 많은 제브라 피쉬 모델은 어른 물고기가 아닌 유충에서 공부, 우리는 겐타 마이신을 사용하여 성인 제브라 피쉬의 옆 선 회생 분석 (이전에 zebrafish의 유충에 사용되는 아미노 글리코 사이드 개발에 초점을 맞추고 분석이 가능했던 있도록 최근에) 성인 물고기 (17)에 사용되는 현재의 성인 zebrafish의 질병 모델에 적용 할 수 있습니다. 이전에 반 트럼프 전자에 의해 절차를 발표하면서t AL. 17 성어 헤어 셀 절제를위한 조건을 확립, 그들은 제어와 같은 형질 전환 지브라 피쉬 라인 또는 약리학 적으로 유도 된 질병 상태를 사용할 때와 같은 실험 그룹 간의 정량적 비교를 위해 필요한 neuromast 재생을위한 표준 곡선을 확립하지 않았다 제브라 피쉬 18. 따라서 우리는 제어 및 실험 그룹을 비교할 때 같은 성인 zebrafish의 질병 모델과 마찬가지로 우리의 데이터를 사용할 수 수사관을 가능하게 neuromast 재생의 표준 곡선을 설정하는 머리 세포 제거에 대한 반 트럼프 등. 17의 절차를 따랐지만 자신의 작품에 구축 . 분석은 또한 해상도가 높은 레벨이 요구 될 때 개별 머리카락 세포로 분석의 확장을 허용하도록 설계되었다.

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

때문에 사람의 청각과 균형 장애의 임상 적 중요성에, 같은 제브라 피쉬와 같은 모델 생물은 옆 선 개발과 재생을 연구하는 데 사용되었습니다. 제브라 피쉬는 그것의 빠른 개발 시간과 높은 재생 능력의 이러한 연구에 특히 매력적이다. 지금까지, 옆 선 중생의 제브라 피쉬의 연구는 주로하기 때문에이 단계에서 neuromasts의 낮은 숫자의 배아와 유생 단계의 물고기를 활용했다. 이 측선 재생 /과 또는 이전 개발 단계에서보다 쉽게​​ 개발의 정량 분석​​을했다. 신경과 비 신경 질환의 많은 제브라 피쉬 모델은 어른 물고기가 아닌 배아 / 유충에서 공부하고 있기 때문에 분석이 가능했던, 그래서 우리는 현재에 적용 할 수있는 성인 제브라 피쉬의 양적 측면 라인 회생 분석 개발에 집중 성인 zebrafish의 질병 모델. 반 TRUM에 의해 이전의 연구에 구축P 등. 17 성인 멕시코 장님 동굴 물고기와 제브라 피쉬 (다니오 rerio)에있는 유모 세포의 제거를위한 절차를 기술 한, 우리의 분석은 제어 및 실험 그룹 사이의 정량적 인 비교를 할 수 있도록 설계되었습니다. 이것은 측선의 규정 된 영역에 모발 세포의 겐타 마이신 유도 된 괴사를 다음 24 시간 기간 동안 neuromast 재현의 퍼센트에 근거 회생 neuromast 표준 곡선을 개발함으로써 달성되었다. 분석은 또한 해상도의 높은 수준이 요구되는 개별 모발 세포 레벨의 분석을 확장 할 수 있도록 설계되었다.

성인 제브라 피쉬의 측면 라인의 neuromast 재생을 정량화하기위한 절차의 최적화. 애벌레 제브라 피쉬의 neuromasts 쉽게 가늠할 수있다; 그러나, 성인 제브라 피쉬의 측면 라인은 6,17,19,20 정량적 분석을 더욱 어렵게 만드는 스티치 당 neuromasts 훨씬 더 많은 수 있습니다. 도 1a에 도시 된 바와 같이, 헤드는 중앙부 또는 꼬리 하나에 비해 neuromasts의 ...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

신경 및 비 신경 질환의 많은 지브라 피쉬의 모델 성어보다는 배아 / 유생으로 연구되어 있기 때문에, 우리는 성인 zebrafish의 질병 모델에 적용 할 수있는 양적 측면 라인 회생 분석법을 개발 하였다. 분석은 1) neuromast 2) 개별 머리 세포 수준의 해상도를하고있었습니다.

성인 Zebrafish의의 측면 라인 재생을위한 분석

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K 조회수.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science.  신경 및 비 신경 질환의 많은 지브라 피쉬의 모델 성어보다는 배아 / 유생으로 연구되어 있기 때문에, 우리는 성인 zebrafish의 질병 모델에 적용 할 수있는 양적 측면 라인 회생 분석법을 개발 하였다. 분석은 1) neuromast 2) 개별 머리 세포 수준의 해상도를하고있었습니다. 

비디오: An Assay for Lateral Line Regeneration in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

성인 Zebrafish의 전체 신장 마로우의 이식

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

연구

생물학

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

MCF - 7 세포주에서 세포 독성 Nanogenomedicine의 : MTT 분석

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

성인 Zebrafish에서 레트로 - 궤도 분사

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

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 ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

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 ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

성인 얼룩말 피시를위한 생체 내 표면 심전도

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...