이 작품은 MCM과 TG에 미국 암 협회 박사 과정 이수 원정대 # PF - 05-041 - 01 - DDC에 NIH R01 부여 HD050901에 의해 지원되었다.

<ol><li> 남성 zebrafish는 여성 물고기 다음 먼저 해부하는 것입니다. 절개를 시작하기 전에 0.2 % Tricaine에서 물고기를 마취 후 15 분 동안 얼음물에 부화하여 안락사. </li><li> 가볍게 종이 타월에 마른 생선을 끝날하고 해부 매트에 배치하여 시작합니다. 외부, zebrafish 단일 지느러미, 꼬리와 항문 지느러미와 결합하여 가슴과 골반 지느러미 (그림 1)가. <img src="/files/ftp_upload/1717/1717fig1.jpg" alt="그림 1" /> 그림 1. 표시된 지느러미 달린 성인 남자 생선. </li><li> 꼬리의 다육 질의 부분을 통해 해부 매트에 생선과 안구 소켓의 복부 부분을 핀. </li><li> 항문 지느러미에 단지 앞쪽에 물고기의 뱃속에있는 피부를 싹둑. 항문 지느러미에서 operculum에 배꼽을 따라 피부와 근육 기본 (아가미를 통해 하드 커버) (그림 2, 1 단계)을 잘라 버릴거야. <img src="/files/ftp_upload/1717/1717fig2.jpg" alt="그림 2" /> 그림 2. 내장을 폭로하기 위해 피부 및 신체 벽면 근육을 제거하는 단계가. </li><li> 다음, 가슴 거들 (지느러미의 기지에서 두께, 뼈다귀 지역) (그림 2 단계 2)를 포함하여 operculum와 가슴 지느러미를 제거합니다. 아래 항문 지느러미 (그림 2, 3 단계)에 다음 생선의 측면을 따라 지금 노출된 길 뒤로 위쪽에서 피부와 근육 기본 시작 컷합니다. </li><li> 조심스럽게 물고기의 측면에서 피부와 기본 근육을 제거하십시오. 내부 장기의 대부분은 (그림 3) 지금은 볼 수 있습니다. <img src="/files/ftp_upload/1717/1717fig3.jpg" alt="그림 3" /> 그림 3. 신체 벽 근육과 성인 남성이 물고기는 내부 장기의 시각화를 허용 제거. </li><li> testes는 등의 신체 벽에 연결된 긴, 백색, 이점 기관입니다. testis를 제거하고 PBS 한 접시에 놓으십시오. spermatagonia에서 spermatids로 개발 배아 세포의 다양한 단계 (릴 외., 2009)와 cysts가 들어있는 정액을 운반하는 tubules (그림 4)를 시각화하는 반사광과 testis을 검사합니다. <img src="/files/ftp_upload/1717/1717fig4.jpg" alt="그림 4" /> 그림 4. testis의 해부. 하얀 둥그스름한 구조는 정액을 운반하는 tubules 수 있습니다. </li><li> 다음 물고기의 뱃속에서 위장 시스템을 제거합니다. 간장은 그 큰 크기, lobed 형태, tannish 색상 및 광범위한 vascularization에서 확인할 수 있습니다. 밝은 빨간색으로 나타납니다 담낭, 녹색 반투명​​ 액체 가득한 색, 그리고 비장은, 내장 내에 볼 수 있습니다. </li><li> 장기의 나머지 부분에서 대장을 분리하고 그것을 스트레칭. 대장의 앞쪽에, 중간 및 사후 지역은 상피 주름의 높이 (월러스 외., 2005)에 의해 정의됩니다. </li><li> PBS에서 소장의 조각을 장소와 전달 빛을 사용하여 상피 주름을 관찰. </li><li> 다음, 수영 방광을 확인합니다. 수영 방광 Weberian 장치 (피니 외., 2006)을 통해 내이 (内耳)에 연결되어있는 공압 덕트, 그리고 앞쪽에 챔버를 통해 식도에 연결된 후부 챔버로 이루어져 있습니다. </li><li> 제거하고 수영 방광을 삭제. 생선을 핀을 빼다 다시 핀 그 복부 쪽 최대 지느러미 몸 벽을 따라있는 신장을 해부하다 수 있습니다. 신장은 지느러미 대동맥과 색소 세포와 관련된 반투명 분홍색 구조입니다. 신장은 머리, 몸, 그리고 꼬리 지역 (그림 5)으로 나뉘어져 있습니다. 신장의 한 부분을 절개하다하고 PBS에 놓으십시오. 신장 tubules을 나타내기 위해 신장 조직을 분리 애타게. <img src="/files/ftp_upload/1717/1717fig5.jpg" alt="그림 5" /> 그림 5. 머리, 몸, 그리고 지느러미 몸 벽을 따라 꼬리 신장의 위치. </li><li> 여성 생선을 해부하다. 이전에 설명한 바와 같이, 얼음 물 속에 물고기를 안락사와 해부 매트에 걸어 건조하기 전에 그것을 두드려. 이전 증명과 같은 물고기의 측면에서 피부를 제거합니다. 난소는 vascularized mesovarium하여 뱃속에 정지하는 bilobed 구조입니다. </li><li> 난소 중 하나 엽을 제거, PBS에 배치하고, 전송 빛을 그것을 검사합니다. oocytes는 좋은 바늘을 사용하여 분리 조롱하고 (그림 6, 셀맨 외., 1993) 개최하실 수 있습니다. 스테이지 I oocytes는 약 10 있습니다 - 크기와 반투명 150 미크론. 크기가 350 마이크론과 대뇌 피질의 과립의 모양에 의해 정의 - 스테이지 II의 oocytes는 약 150입니다. 스테이지 III의 oocytes는 350 있습니다 - 크기가 750 마이크론과 난황의 축적으로 인해 불투명하고 있습니다. 성숙 단계 IV 및 V oocytes는 반투명하고 일반적으로 최근에 짝짓기가 암컷의 난소에서 찾을 수 없습니다. <img src="/files/ftp_upload/1717/1717fig6.jpg" alt="그림 6" /> 그림 6. 라이브 무대 I, II, III 및 제품 oocytes은 아래의 관찰전송 빛을 현미경을 해부. </li><li> 다음 물고기의 마음을 해부하다. 마음은 후부와 아가미에 복부에 위치하고 있습니다. 심장과 주변 조직의 모든을 절단하고 PBS에 배치하여 시작합니다. 조심스럽게 섬세한 아트리움을 손상하지 않도록주의하고, 심장을 둘러싼 조직을 멀리 해부하다. </li><li> 심장 박동을 관찰하기 위해 링어의 솔루션으로 해부하는 마음을 놓으십시오. 아트리움, 뇌실 및 bulbus arteriosus (그림 7, 후진타오 외., 2001)를 식별합니다. <img src="/files/ftp_upload/1717/1717fig7.jpg" alt="그림 7" /> 그림 7. 어른 마음을 해부 했어요. </li><li> 물고기의 머리를 제거하여 절개를 완료합니다. 물고기를 unpinning와 면도날로 머리를 제거하여 시작합니다. 포셉와 두개골의 복부 측면에서 가능한 한 많은 부드러운 조직으로 제거합니다. 작은 스프링 가위를 사용하여 눈을 제거합니다. 두개골을 열고 브레이크와 두뇌의 복부 측면에서 뼈를 제거합니다. 지금은 PBS의 접시에 머리를 장소와 뇌의 등의 측면에서 피부와 두개골 뼈를 제거합니다. </li><li> , telencephalon, habenula, 광 tectum, 소뇌 및 모수 (., 실링, 2002 그림 8, Wullimann 외, 1996) 후각 구근을 확인합니다. <img src="/files/ftp_upload/1717/1717fig8.jpg" alt="그림 8" /> 그림 8. 해부 성인 zebrafish의 뇌. 맨 앞쪽에 도설보기. </li></ol>

<ol>
	<li>Finney, J. L., Robertson, G. N., McGee, C. A., Smith, F. M., Croll, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Autonomic+Innervation+of+the+Swim+Bladder+in+the+Zebrafish+(Danio+rerio).">Structure and Autonomic Innervation of the Swim Bladder in the Zebrafish (Danio rerio).</a> J Comp Neurol. 495, 587-606 (2006).</li><li>Hu, N., Yost, H. J., Clark, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+morphology+and+blood+pressure+in+the+adult+zebrafish.">Cardiac morphology and blood pressure in the adult zebrafish.</a> Anat Rec. 264, 1-12 (2001).</li><li>Leal, M. C., Cardoso, E. R., N&#243;brega, R. H., Batlouni, S. R., Bogerd, J., Fran&#231;a, L. R., Schulz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+stereological+evaluation+of+zebrafish+(Danio+rerio)+spermatogenesis+with+an+emphasis+on+spermatogonial+generations.">Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.</a> Biol Reprod. 81, 177-187 (2009).</li><li>Schilling, T. F., Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+larval+and+adult+zebrafish.">The morphology of larval and adult zebrafish.</a> Zebrafish: A Practical Approach (The Practical Approach Series). , (2002).</li><li>Selman, K., Wallace, R., Sarka, A., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+oocyte+development+in+the+Zebrafish,+Brachydanio+rerio.">Stages of oocyte development in the Zebrafish, Brachydanio rerio.</a> J. Morphol. 218, 203-224 (1993).</li><li>Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+growth+and+differentiation+in+zebrafish.">Intestinal growth and differentiation in zebrafish.</a> Mech Dev. 122, 157-173 (2005).</li><li>Wullimann, M. F., Rupp, B., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy of the zebrafish brain : a topological atlas. , (1996).</li></ol>

동물 실험은 펜실베니아 기관 애니멀 케어 및 사용위원회의 대학에 의해 명시된 지침과 규정에 따라 수행되었다.

시약 : 0.2 % Tricaine (에틸 3 aminobenzoate) 200 MG tricaine 분말 97.9 ML DD 물 ~ 1 ML 1 M 트리스 (산도 9) 7.0 산도를 조정 인산은 생리 (PBS)를 버퍼 4.0 g NaCl 0.1 g KCl 100 ML 0.1 M PO 4 버퍼, pH를 7.3 150 ML DH 2 O 링거스 솔루션 6.7g NaCl 0.2g KCl 0.2g CaCl 2 1.2g의 Hepes 1 LH 2 O 7.2 산도를 조정 장비 : 매트와 해부 접시 전자 현미경 과학 카탈로그 # 70540 Vannas 스프링 가위 파인 과학 도구 카탈로그 # 91500-09

dissection of organs from the adult zebrafish

지난 20 년 동안, zebrafish는 척추 개발과 질병을 이해하는 강력한 모델 생물되었다. 배아 및 유충의 실험 분석 광범위하며 형태가 잘 문서화되어 있지만, 함께 어른과 함께 작업을위한 기술과 성인 zebrafish의 해부학 및 성인 구조와 장기 발전 연구에 대한 설명이 부족합니다. 유충의 기관들은 전체적인 구조, 형태, 그리고 어른 전환하는 애벌레 동안 해부학 위치에 중대한 변화를 받고있다. 내부의 장기는 거대한 성장과 개조를 받아야하는 동안 외부, 투명 유충은 그 특성 성인 줄무늬 안료 패턴과 결합하여 골반 지느러미를 개발하고 있습니다. 또한, bipotential 생식선의 primordium은 testis이나 난소 중으로 개발하고 있습니다. 이 프로토콜은 성인의 기관의 많은 식별하고 두뇌, 생식선, 위장병 시스템, 심장, 그리고 성인 zebrafish의 신장의 해부하는 방법을 보여줍니다. 해부하는 기관은 현장 하이브리드화에 immunohistochemistry, 조직학, RNA 추출, 단백질 분석, 및 다른 분자 기법을 사용할 수 있습니다. 이 프로토콜은 애벌레 기관의 리모델링, 성인 장기 시스템의 성인 및 기타 조사 특정 장기의 morphogenesis을 포함 zebrafish의 연구 확대에 도움이 될 것입니다.

Watch this Scientific Journal Video about Dissection of Organs from the Adult Zebrafish at JoVE.com

Dissection of Organs from the Adult Zebrafish

이 프로토콜은 성인 zebrafish에서 장기를 식별하고 해부를위한 절차를 설명합니다.

성인 Zebrafish에서 기관의 해부

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

dissection-of-organs-from-the-adult-zebrafish

Research

JoVE Journal

Biology

103.9K 조회수.  University of Pennsylvania-School of Medicine. 이 프로토콜은 성인 zebrafish에서 장기를 식별하고 해부를위한 절차를 설명합니다.

비디오: Dissection of Organs from the Adult Zebrafish

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

성인 Zebrafish에서 내이 (内耳) 감각 상피 조직의 총과 파인 해부 ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

연구

생물학

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

성인 마우스 Hippocampal 이가있는 이랑의 해부

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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, ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

성인에서 Oenocytes의 해부 Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

성인 Zebrafish의 신장의 해부

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

애벌레의 심장 해부, 소년과 성인 Zebrafish, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...