FIPE / HCPA - Fundo 드 Incentivo Pesquisa 전자 Eventos 망토 - <a href="http://www.capes.gov.br/">Coordenação 드 Aperfeiçoamento 드 Pessoal 드 Nível 우수</a>

1. 프로토콜 텍스트 <ol><li> zebrafish 혈액을 수집하기 전에 anesthetizing 물을 준비할 필요가 있습니다. 500 ML 용량의 용기에 수족관 물 ~ 200 ML 하거라. 얼음 칩의 ~ 200g을 추가합니다. 온도는 4 ° C.에 대해이어야합니다 얼음 조각이 녹아 때문에, 4 ° C. 가까운 일정한 온도를 유지하는 것이 더 아이스 칩을 추가할 필요합니다 </li><li> anesthetizing 물이 준비되면, 혈액 수집에 필요한 자료를 준비합니다. P20 pipettor에 낮은 유지 팁을 넣고 그것을 쉽게 액세스할 수 pipettor 둡니다. 피펫 팁은 오염에 대한 소스로 연락하도록 허용하지 마십시오. </li><li> 마른 거즈 한 조각과 페트리 접시를 덮어. 강철 칼날과 거즈의 다른 조각이 쉽게 접근할 장소에 배치해야합니다. </li><li> 플라스틱 튜브에 맞게 원심 분리기가 필요합니다. </li><li> 상기 자료를 준비하면 anesthetized 재치로 첫 번째 zebrafish를 캡처헥타르 낚시 그물과 마취에 대비되어 물속에 그것을 풀어주고있다. Zebrafish는 차가운 물에 3-6의이 물고기에 따라 anesthetized해야합니다. 그것이 더 이상 외부 자극에 응답 없을 때까지 차가운 물에 물고기를 유지. </li><li> 낚시 그물을 사용, 거즈의 꼬리를 떠나, 거즈의 준비된 조각에 anesthetized 물고기를 넣습니다. 오직 꼬리를 빠뜨린 생선의 머리와 몸 위에 거즈를 접으십시오. 배양 접시에 거즈로 덮여 생선을 넣어. </li><li> 단지 항문 지느러미와 꼬리 지느러미 사이에 대각선 절개를 만들기 위해 강철 칼날을 사용합니다. 피가 나올 시작합니다. 이 시점에서 그것은 신속하게 일할 필요가 있습니다. </li><li> 부드럽게는 P20의 pipettor (낮은 유지 팁과 함께 미리로드)와 함께 나오는 혈액을 열망. 수집될 수있는 혈액의 양은 생선의 크기에 무엇을 제대로 anesthetized 것을 어느 정도까지 달려있다. 그것은 보통 5 ~ 20 μl에 따라 다릅니다. 혈액은 C를 멈추면아웃 oming, 부드럽게 튜브로 했잖아요 혈액을 전송합니다. </li><li> 용혈을 피하기 위해, 그것은 원심 분리기에 배치 될때까지 피가 묻어있는 튜브가 어떤 과감한 움직임없이 매우 신중하게 처리하는 것이 중요합니다. </li><li> 용혈을 피하기 위해서는 혈액 샘플은 혈액의 10 분 이내에 원심 분리기에 확보하는 것이 또한 중요합니다. </li><li> 필요하다면, 그것은 수영장을 만드는 하나 이상의 동물에서 혈액을 결합할 수 있습니다. 풀링된 샘플은 첫 번째 물고기와 원심 분리의 혈액 사이의 지연 시간이 10 분을 초과하지 않는만큼 잘 동작합니다. </li><li> 혈액 수집이 완료되면, 0.5 G (Eppendorf의 원심 5415D)에서 10 분 동안 혈액을 원심 분리기. </li><li> 원심 분리 후, 혈청 튜브의 상단 레이어에 있습니다. 피펫으로 두 레이어가 잘 나누어하고 안정적​​인 유지하면서 단지 혈청을 받아보실 만드는 혈청 기음. </li><li> 새로운 microtube에 혈청을 전송하고생화 학적 분석에 사용될 준비가되어 있습니다. 이것은 생화 학적 분석을 시작 기다리는 동안 혈청은 얼음에 저장할 수 있습니다. </li><li> 혈청은 즉시 사용하지 않을 경우에는, 약 3 개월 -18시 ° C에 냉동 수 있습니다. </li></ol> 2. 대표 결과 그것은 이전에 설명한 기법 (표 2)보다 4 배 더 많은 혈액을 상징하는 그 무엇을 각 생선 전체 혈액의 5-20 μl를 수집하는 것이 가능했습니다. 총 콜레스테롤, HDL-콜레스테롤, LDL-콜레스테롤, 트리 글리세 리드의 생화 학적 및 분석이 기법을 사용하여 혈액 후에 수행되었다. 혈액 수집은 식품 섭취량의 간섭을 피하기 위해 전에 남녀 물고기 두 그룹은 24 시간 동안 금식했습니다. 분석은 총 콜레스테롤 및 트리 글리세 라이드 분석을위한 작은 비늘 colorimetric 테스트 (Labtest Diagnóstica SA, 브라질) 다 있었는데, 혈청 중 3 μl가 사용되었습니다. LDL-콜레스테롤과 HDL-콜레스테롤의 경우분석, 4 μl 및 혈청 10 μl는 각각 사용 하였다. 이러한 분석은 시료 당 10 zebrafish의 풀링된 샘플에서 수행되었다. 세럼 lipidic 수준은 자신의 계란과 하단 커버 수족관에서 2 주 실험 기간 동안 자신의 달걀에 대한 액세스 권한이없는거야, 이들을 액세스할 생선 사이에 비교했다. 세럼 분석 보여주 그 혈청 총 콜레스테롤 수치 (계란 362 ± 42 밀리그램 / DL와 계란 357없이 ± 13 밀리그램 / DL), HDL-콜레스테롤 (달걀 91.22 ± 1.79 밀리그램 / DL과 계란 72.14없이 ± 2.89 밀리그램 / DL) 및 (계란 55.68 ± 10.88 MG / DL과 계란 44.18 ± 9.84 밀리그램 / DL 제외) LDL-콜레스테롤은 그룹간에 크게 다를하지 않았다. 그러나, 트리 글리세 라이드 수준은 (; P = 0.03 달걀 457 ± 25 밀리그램 / DL 포함) 컨트롤 그룹에서보다 (계란 292 ± 64 밀리그램 / DL없이) 실험 그룹에서 상당히 낮은 있었다. <table border="1" ><tbody><tr><td> &amp; NBSP; </td><td> 계란에 접근할 수있는 </td><td> 계란에 액세스하지 않고 </td></tr><tr><td> 총 콜레스테롤 (MG / DL) </td><td> 362.82 ± 73.11 </td><td> 357.69 ± 23.08 </td></tr><tr><td> LDL - 콜레스테롤 (MG / DL) </td><td> 55.69 ± 18.84 </td><td> 44.19 ± 17.05 </td></tr><tr><td> HDL - 콜레스테롤 (MG / DL) </td><td> 91.23 ± 3.11 </td><td> 72.14 ± 5​​.01 </td></tr><tr><td> 트리 글리세 리드 (MG / DL) </td><td> 457.64 ± 43.78 * </td><td> 292.36 ± 111.28 </td></tr></tbody></table> 표 1. 두 연구 그룹에 대한 콜레스테롤과 트리 글리세 리드 seric 수준 (계란에 대한 액세스 권한이있는 계란에 대한 접근없이) 평균 ± 표준 편차로 표현. * 통계적으로 의미 (P = 0.03). 학생 t 시험. <table border="1"><tbody><tr><td> AUT<html> hors </td><td> 절개의 장소 </td><td> 수확 방법 </td><td> 마취 </td><td> 수집된 혈액의 금액 </td></tr><tr><td> Jagadeeswaran 동부 표준시. 알., 1999 Murtha 외., 2003 </td><td> 지느러미 지느러미에 미세 절개의 후부 </td><td> Micropipette </td><td> 언급되지 차가운 물에 MS222 3퍼센트 </td><td> 한 5 μl 5 10 μl </td></tr><tr><td> Eames 외., 2010 </td><td> 목베기 </td><td> 마이크로 모세관 튜브 </td><td> MS222 0,02 % 28 ° C 물 </td><td> 5 10 μl </td></tr><tr><td> 현재의 연구 </td><td> 항문 지느러미와 꼬리 지느러미 사이에 절개 </td><td> Micropipette과 낮은 유지 도움말 </td><td> 물과 얼음 칩 </td><td> 5 20 μl </td></tr></tbody></table>앞에서 설명한 혈액 수집 기술과 현재의 연구에서 설명 한 사이 e_content &quot;&gt; 표 2. 비교.

<ol>
	<li>Schneider, A. C. R., dos Santo, J. L., Porawski, M., Schaefer, P. G., Maurer, R. L., Matte, U., da Silveira, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementa&#231;&#227;o+de+um+novo+modelo+de+experimenta&#231;&#227;o+animal+Zebrafish.">Implementa&#231;&#227;o de um novo modelo de experimenta&#231;&#227;o animal Zebrafish.</a> Rev. HCPA. 29, 100-103 (2009).</li><li>Briggs, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish:+a+new+model+organism+for+integrative+physiology.">The zebrafish: a new model organism for integrative physiology.</a> Am. J. Physiol. Regulatory Integrative Comp. Physiol. 282, 3-9 (2002).</li><li>Menke, A. L., Sptsbergen, J. M., Wolterbeek, A. P. M., Woutersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+anatomy+and+histology+of+adult+Zebrafish.">Normal anatomy and histology of adult Zebrafish.</a> Toxicologic Pathology. 000, 1-16 (2011).</li><li>Murtha, J. M., Qi, W., Keller, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematologic+and+serum+biochemical+values+for+Zebrafish.">Hematologic and serum biochemical values for Zebrafish.</a> Comp. Med. 53, 37-41 (2003).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br. J. Haematol. 107, 731-738 (1999).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol. Dis. 25, 239-249 (1999).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7, 205-213 (2010).</li><li>Kilpatrick, E. S., Rumley, A. G., Rumley, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+haemolysis+on+blood+glucose+meter+measurement.">The effect of haemolysis on blood glucose meter measurement.</a> Diabet. Med. 12, 341-343 (1995).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Organs+from+the+Adult+Zebrafish.">Dissection of Organs from the Adult Zebrafish.</a> J. Vis. Exp. (37), e1717 (2010).</li><li>Kinkel, M. D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+Injection+into+Adult+Zebrafish.">Intraperitoneal Injection into Adult Zebrafish.</a> J. Vis. Exp. (42), e2126 (2010).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+Injection+in+Adult+Zebrafish.">Retro-orbital Injection in Adult Zebrafish.</a> J. Vis. Exp. (34), e1645 (2009).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

본 논문은 zebrafish 실험에 더 이상 혈액 및 혈청 분석을 수있는 간단한 방법을 제공합니다. 이 기술은 혈액 매개 변수 데이터를 필요로하는 미래의 zebrafish hematologic 연구에 기여할 수있는 잠재력을 가지고 있습니다. 그것은 또한 실험적인 모델로서 zebrafish의 큰 어플 리케이션을 위해 허용해야합니다. 이 기술은 특별한 기술이나 정밀한 기술의 구현을 필요로하지 않습니다. 또...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 낮은 유지 팁 </td><td> 응용 Biosystems </td><td> 022,493,020 </td><td></td></tr><tr><td> Eppendorf의 원심 분리기의 5415D </td><td> Eppendorf </td><td> 중단 </td><td></td></tr></tbody></table>

blood collection for biochemical analysis in adult zebrafish

zebrafish는 여러 인간의 질병 연구를위한 동물 모델로 사용되었습니다. 그것은 분자 이벤트 및 치료 전략의 공부뿐만 아니라 일부 pathologies 1의 생리적 메커니즘을 평가하기위한 강력한 잠복기 플랫폼으로 검색할 수 있습니다. 연구자들이 zebrafish 시스템의 탐사를 허용하는 데 필요한 기본적인 기법 3 부족한 것을 추측하는 위험성이있다 장기 및 시스템 2의 성인 zebrafish의 생리학에 관련된 상대적으로 몇 가지 간행물이 있습니다. zebrafish의 Hematologic 생화학 값은 첫 번째 1999 년 Jagadeeswaran 및 동료에 의해 ​​기술된 혈액 수집 기술을 채용 Murtha 및 동료의 사에 의해 2003 년에보고되었다. 간단히, 혈액은 길이 약 0.3 cm는 지느러미 대동맥 5 지역에서, 가로 절개를 통해 micropipette 팁 통해 수집되었다. 참여하는 순간 차원이 때문에고도로 숙련된 실천을 요구하는 고정밀도의 기술이다. 동일한 기술은 같은 해에 6에있는 다른 간행물에 동일한 그룹에 의해 사용되었다. 2010 년에는 Eames와 동료 zebrafish 7 전체 혈당 수준을 평가. 그들은 가위로 해고당하는 것까지을 수행하고 가슴 분절로 heparinized microcapillary 수거 튜브를 삽입하여 혈액에 접속할 수있었습니다. 그들은 작품 Kilpatrick 외 토대로 적절한 보관 온도로 해결되었다 용혈 어려움을 언급. 8. 저희 연구실에서 Jagadeeswaran의 기술을 사용하려고 시도하면, 우리는 모음이 시작되기 전에 손실되는 혈액의 상당량을 허용하지 않기로 정확하게 올바른 장소에 절개를 만들기 위해 어려운 것으로 나타났습니다. 최근 굽타 외 9.는 성인 zebrafish 기관, Kinkle 외 해부하는 방법을 설명했다. 10 intraperiton을 수행하는 방법을 설명eal 주사, 그리고 Pugach 외. 11 역전사 궤도 주사를 수행하는 방법을 설명했다. 그러나, 더 많은 작품은보다 완벽하게 zebrafish 연구를위한 기본적인 기술을 탐구하는 데 필요합니다. zebrafish의 작은 크기는 실험 모델로 이용하여 연구자에 대한 도전을 선물한다. 또한 규모의이 소 주어, 그것은 단순한 기술이 연구자들이 zebrafish 모델의 장점을 탐험 수 있도록 개발된 것이 중요합니다.

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

Blood Collection for Biochemical Analysis in Adult Zebrafish

본 논문은 Zebrafish의 지느러미 대동맥에서 혈액을 수집하는 기술을 선물한다. 그것은 또한 콜레스테롤과 트리 글리세 라이드 수준을 결정하기위한 시험과 같은 생화 학적 분석에 사용을 위해 혈청을 얻기위한 지침을 제공합니다.

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 ...

blood-collection-for-biochemical-analysis-in-adult-zebrafish

Research

JoVE Journal

Biology

36.2K 조회수.  Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental. 본 논문은 Zebrafish의 지느러미 대동맥에서 혈액을 수집하는 기술을 선물한다. 그것은 또한 콜레스테롤과 트리 글리세 라이드 수준을 결정하기위한 시험과 같은 생화 학적 분석에 사용을 위해 혈청을 얻기위한 지침을 제공합니다.

비디오: Blood Collection for Biochemical Analysis 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

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish의 두뇌 뇌실 주입

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

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 ...

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 ...

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 ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

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 development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

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 ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

성인 Zebrafish의 신장에서 네프론의 구성 및 기능 분석

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

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 ...

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).