마리 Birne, Areti Tsiola, Jaymie 에스테베즈 및 Arelys 우리베 감사합니다. 이 작품은 CUNY 연구 재단과 학부생을위한 하워드 휴즈 의학 연구소 부여 여름 프로그램 (평)와 NIH RO3 41702-00-01에 의해 부분적으로 지원되었다. 연구는 또한 퀸즈 대학과 실험의 일부 이미징, 퀸즈 대학에서 세포와 분자 생물학의 핵심 시설의 설비에서 수행되었다에 의해 지원되었다.

Zebrafish는 28에서 퀸즈 대학 동물 시설 13 표준 프로토콜 ° C.를 사용하여 제기되었습니다 개인 생선 쌍 또는 그룹 matings은 preformed하고 그 결과 태아는 청소와 28 ° C 인큐베이터에 보관해야합니다. 물고기의 건강은 매일 검사해야합니다. 5 일 포스트 수정 (dpf)에서, 물고기는 물고기 10-15 밀도에서 탱크로 구분해야하며 생선 보육 시설에 들어갔다. 15, 30, 45, 60, 90, 180 dpf : 여기에서 제시한 프로토콜에 대한 zebrafish는 성인 단계를 통해 유충에서 수집된 있었다. 1. Zebrafish 제목 수집 및 고정 <ol><li> 마취로 0.2 % Tricaine에 그물과 장소를 사용하여 서식 탱크에서 물고기를 제거합니다. </li><li> 안락사에 15 분 동안 얼음물에 anesthetized 물고기를 배치합니다. </li><li> 1X에 신선한 4 % paraformaldehyde (PFA)를 확인 인산 버퍼 식염수 (PBS)를. </li><li> 4 다음 하룻밤 RT에서 1~2시간에 대한 4% PFA 수집 물고기를 품어 ° parafilm에 싸여 배양 접시에있는 C. 이것은 물고기 평면을 유지하고 나중에 절개가 훨씬 쉬워집니다. </li><li> 고정 후, 십대 초반 (PBT)로 두 번 PBS로 씻어. 물고기는 최대 10 일 4 ° C에서 PBT로 유지하실 수 있습니다. </li></ol> 2. 물고기와 절개를 측정 <ol><li> 페트리 접시의 절반에 장소 하나의 생선은 신선한 PBS 가득한과 그 오른쪽에 그것을 믿는다. </li><li> 아니라 디지털 mm 캘리퍼스 (그림 1)를 사용하여 꼬리 지느러미를 포함하여 주둥이에서 꼬리 자료 (꼬리 꽃자루)에 물고기의 측정 길이. 이것은 표준 길이 (SL)입니다. </li><li> 각 생선 번호 / 문자 조합을 나중에 분석을 위해 추적하는 개인 생선과 결과를 해부 심장을 허용을 지정할 수 있습니다. 부모 재고 번호, 고정, SL 측정에 나이를 포함하여 데이터, 스프레드 시트에 입력되었으며 이러한 레이블을 기반으로 조직. </li><li> 해부, 라벨 PCR 튜브 지정된 추적 번호와 스트립 및 PBT 또는 추가 분석을 위해 적절한 다른 솔루션을 작성하기 전에. </li><li> 배양 접시에있는 동양 물고기 복부 측면까지 눈과 아가미 (그림 2) 사이의 고개를 들고 집게로 시신을 안정화하면서. </li></ol> 작은 물고기 (12mm SL보다 작은) : <ol start="4"><li> A) 핀 홀더에 날카로운 포셉 또는 마이크로 바늘을 사용하여 마음을 나타내기 위해 신체의 가슴 근육과 지느러미를 제거합니다. 해부 동안 포셉의 끊임없는 움직임은 물고기를 이동 PBT에 전류를 발생할 수 있습니다. 이것은 따라서 마이크로 바늘이 가슴 근육과 지느러미를 제거하고 뱃속을 열고하는 데 사용할 수있는 작은 생선, 특히 문제가됩니다. </li><li> A) 부드럽게 중앙홀 아래에서 구멍의 마음을 밖으로 치우도록 포셉을 사용합니다. 반면, 일부 생선에 부드럽게 주먹코 밖으로 당기하고 마음의 나머지는 따를하여 마음을 제거할 수 있습니다. 쉽게 손상될 수 있으므로이 기술을 사용하는 경우 아트리움의 위치에주의하십시오. 괜찮 후 삭제 될 수있는 마음, 함께 제공 추가 조직이있다면. </li><li> 물고기 같은 TG (myl7로 형광등 심장 마커가 포함된 경우) : GFP) twu34를 해부를위한 형광 범위를 사용하여 개화 (그림 3)에 의해 심장의 제거를 확인합니다. </li><li> 마음 한번)가 구멍에서 제거, 심장의 외부에서 추가가 아닌 심장 조직 및 epicardial 안감을 제거하기 위해 마음과 마이크로 바늘을 개최 포셉을 사용합니다. </li></ol> 큰 물고기 (12mm SL보다 큼) <ol start="4"><li> 아가미 2 일까지 가로자를 - - 앞쪽에 배를 가로 3 컷 - 모두 상처를 연결하는 복부 측면에 화살 컷 (그림 4) 1 : B) 처리 마이크로 스프링 가위를 사용하여 1mm 깊이보다 세 incisions합니다. </li><li> B) 날카로운 집게를 사용하면 뱃속을 열고 심낭의 은빛 조직 (그림 5A)을 나타내기 위해 신체의 가슴 근육과 지느러미를 제거합니다. 이 pericardial 조직을 제거하고 심장 (그림 5B) 표시가 될 것입니다. </li><li> B) 심장에 뛰어난있는 주먹코 arteriosus에 연결된 동맥을 잘라 마이크로 가위를 사용합니다. 이 동맥이 절단되면 아트리움에 따라 포셉 팁을 넣어, 캐비티의 마음을 밖으로 치우도록 포셉을 사용합니다. 괜찮아요 나중에 제거할 수있는 마음, 함께 제공 추가 조직이있다면. </li><li> 심장이 구멍에서 제거되면 B), 마음을 가질 두 집게를 사용하여 마음을 가질 추가 조직, 또는 포셉 한 쌍을 제거하고 마이크로 바늘 추가가 아닌 심장 조직을 제거합니다. 부드럽게 조직을 근근이 살아가고하여 원하는 경우 그 검은 착색에 의해 분명 epicardial 안감은, 심장의 외부에서 제거할 수 있습니다. </li></ol> 3. 마음의 사진 <ol><li> 23g / L 영양 한천 배양 접시를 준비하고 PBS로 확정 한천을 커버. </li><li> 포셉 사용하여 심장이 사진 촬영을 위해 원하는 방향으로 나머지 수있는 한천에서 잘합니다. </li><li> Remov단단히 포셉과 구상의 끝을 잡고하여 PCR 튜브에서 E는 심장. 한천 요리에 마음을 놓으십시오. </li><li> 사진에 한천에 동양의 마음. 예제는 가능한 모든 방향 (그림 6) 각 사진 뒤에 회전 수 있습니다. </li><li> 마음을 사진에 오버헤드 가볍고 짧은 노출 시간을 사용합니다. 최고의 사진을 얻기 위해 필요한 노출을 사용하여 빛을 품질에 대한 조정을합니다. </li></ol> 4. 대표 결과 : 잘 해부 깨끗한 마음의 예제는 그림 6에 여러 방향으로 표시됩니다. 각 심장 전체 형태에서 약간의 차이가되지만 마음은 그대로 뇌실, 아트리움과 주먹코 arteriosus가 포함된 완료해야합니다. 전체 형태에 변화의 몇 가지 예제는 그림 7에 표시됩니다. 까만점 epicardial 조직도 마음에서 제거하실 수 있습니다. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="그림 1"/> 그림 1. 주둥이에서 디지털 mm 캘리퍼스를 사용하여 꼬리 꽃자루에 생선을 측정. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="그림 2"/> 그림 2. 눈과 아가미 사이 포셉로 물고기를 잡아. <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="그림 3"/> 그림 3과 같은 TG와 같은 심장 특정 형광 유전자 변형 마커 (myl7 : GFP). 여기에 표시 twu34은, 작은 생선을 해부하는 마음을 시각화에 도움이 있습니다. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="그림 4"/> 그림 4 가슴 근육과 피부를 제거하는 세 incisions : 1 - 아가미 2 일까지 가로 절단 - 앞쪽에 배를 가로 3 컷 - 모두 상처를 연결하는 복부 측면에 화살 했네요.. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="그림 5"/> . 주먹코 arteriosus (BA), 뇌실 (V), 그리고 아트리움 (A) : 그림 5 블랙 문자는 심장의 실 레이블을. - 상체 구멍은 심낭의 은빛 조직을 보여주 개방됩니다. B - 후 심낭을 제거, 마음이 보이는 쉽게 제거됩니다. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="그림 6"/> 마음의 그림 6. 다른 방향이 표시됩니다. 주먹코 arteriosus (BA), 뇌실 (V), 그리고 아트리움 (A) : 블랙 문자는 심장의 실 레이블을. 앞쪽에 (A), (D) 지느러미, (P) 사후, 복부 (V), 왼쪽 (L)과 오른쪽 (R) : 회색 화살표는 위치와 방향의 중심 본문에 정상적인 위치에 대한 언급을 나타냅니다. 스케일 바는 400μm 나타냅니다. 심장의 A - 복부보기 마음의 B - 지느러미보기 왼쪽에서 C - 가로보기 오른쪽에서 D - 가로보기 <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="그림 7"/> 그림 7. 다양한 크기의 마음의 형태론가 표시됩니다. 주먹코 arteriosus (BA), 뇌실 (V), 그리고 아트리움 (A) : 블랙 문자는 심장의 실 레이블을. 스케일 바 400 μm의를 지적했다. 이하 12mm 이상 SL과 AC - 하트 양식 물고기 12mm보다 큰 SL와 DF - 하트 양식 물고기

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관심 없음 충돌 선언하지 않습니다.

포스트 배아 물고기의 zebrafish 심장의 해부에 대한 이러한 방법은 손상되지 않은 마음의 완전한 제거를 허용합니다. 이 기술은 간단하고 마음을 손상하지 않도록 할 외부 상처에 대한 가이드를 제공하고 있지만, 꾸준한 손 작은 물고기를 위해 필수적입니다. 해부 중 주요 단계는 마음이 골목에서 직접입니다로 은빛 심낭을 식별하는 것입니다. 심낭을 떠올리면 뱃속을 찾는하...

<table border="1"><tbody><tr><th> 자료 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> Paraformaldehyde </td><td> EMD </td><td> PX0055 </td><td> PBS에 .04g/mL </td></tr><tr><td> 집게 # 55 </td><td> FST </td><td> 11295-51 </td><td></td></tr><tr><td> 마이크로 가위 </td><td> FST </td><td> 15005-08 </td><td></td></tr><tr><td> 핀 홀더 </td><td> FST </td><td> 26016-12 </td><td></td></tr><tr><td> 마이크로 바늘 </td><td> FST </td><td> 10130-10 </td><td></td></tr><tr><td> 영양 한천 </td><td> BBL </td><td> 11,472 </td><td> 23g / 소주 H 2 O에 L </td></tr><tr><td> 배양 접시 </td><td> BD 팔콘 </td><td> 351029 </td><td></td></tr><tr><td> PCR 8 관 스트립 </td><td> 미국 과학 </td><td> 1402년부터 2500년까지 </td><td></td></tr><tr><td> 디지털 캘리퍼스 </td><td> 피셔 과학 </td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

Zebrafish는 배아 심장 개발 연구 (최근 리뷰 1-6 참조) 유익하고 실용적인 모델 생물되고있는 그러나, 7-10을 제한되었습니다 성인 심장 개발을 통해 이후의 배아 검사 작동합니다. 성숙과 노화 마음의 변화 형태를 검사하는 것은 청소년과 어른 마음을 준비하고 분리에 사용할 기술의 부족에 의해 제한됩니다. 물고기의 수명 동안 심장 개발을 분석하기 위하여, 우리는 수많은 단계에서 zebrafish 마음을 해부하다하고 자세한 분석 11 그들을 사진. 심장의 형태학의 기능을 쉽게 계량 할 수 있으며, 개인의 마음은 더욱 표준 방식의 호스트에 의해 분석하실 수 있습니다. Zebrafish는 변수 속도로 성장하고 성숙 물고기 성숙의 계기로 포스트 고정, 우리 사진 및 측정 물고기의 길이, 따라서, 나이보다 생선 크기가 더 연결합니다. 이 프로토콜은 30mm와 VL 1mm의 SL 또는 더 큰와 어른 100μm 뇌실 길이 (VL)의 마음으로 애벌레 3.5mm 표준 길이 (SL)에 이르기까지 zebrafish에 대한 두 개의 크기에 의존 해부 기술을, 설명합니다. 애벌레와 어른 물고기는 아주 독특한 신체와 장기의 형태 있습니다. 애벌레는 훨씬 작습되지 않습니다, 그들은 덜 안료를 각 기관은 시각적으로 파악하는 것은 매우 어렵습니다. 이러한 이유로, 우리는 뚜렷한 해부 기술을 사용합니다. 우리가 euthanization 후 직접 해부하는 마음은 다음 고정을 제거 마음에 비해 심방과 같은 매우 느슨한과 풍선과 함께,보다 변수 형태를 갖고 발견으로 우리는 사전 해부 고정 절차를 사용합니다. 물고기는 생체내의 형태와 챔버 위치 (데이터가 표시되지 않음)에 보유, 이전 절개로 고정. 또한, 데모의 목적을 위해, 우리는 심장 (심근) 특정 GFP 유전자 변형 TG (myl7 : GFP)을 활용하여 우리가 전체 마음을 시각화 수 있으며 개발 초기 단계에서 특히 유용합니다 twu34 (12), 때 심장 형태 주변 조직에서 적은 구분됩니다. 심장의 해부는 현장 하이브리드화, immunohistochemistry, RNA 추출하거나 사후 배아 zebrafish에서 쉽게 다른 분석 방법을 사용하여 심장 개발과 성숙을 기본 세포와 분자 생물학의 추가 분석을합니다. 이 프로토콜은 심장 개발 성숙과 노화 연구에 대한 귀중한 기술을 제공합니다.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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

여러 발달 단계에있는 zebrafish 심장의 해부 및 격리에 대한 명확하고 표준화된 방법 설명되어 있습니다. 주석과 부량 기법도 설명합니다.

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 ...

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Research

JoVE Journal

Biology

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

21.3K 조회수.  Queens College, City University of New York. 여러 발달 단계에있는 zebrafish 심장의 해부 및 격리에 대한 명확하고 표준화된 방법 설명되어 있습니다. 주석과 부량 기법도 설명합니다.

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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila Melanogaster에서 애벌레 CNS의 해부

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

연구

생물학

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila 애벌레 NMJ의 해부

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

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 ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

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 and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

해부 그리고 스테 이닝 Drosophila 애벌레 난소

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

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 ...

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 ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Zebrafish의 일반 관리 및 유지 관리 (<em&gt; Danio rerio</em&gt;) 실험실 : 소개

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Danio rerio를 사용하여 병적 인 인간 게놈의 생체 모델링에

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

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 ...