이러한 방법은 지원이 드폴 대학 연​​구위원회와 JT (DE016678)에 NIH / NIDCR R01 부여에 의해 제공되었다 JT의 연구실에서 연구 휴가 동안 장어에 의해 개발되었습니다. 우리는 기꺼이 캐롤라인 헌터, CMRC의 zebrafish 핵심 시설에 동물 보호 기술자와 폴리 Pawluczuk 우리 학부 실험실 조교의 노력을 인정합니다.

Zebrafish 축산 Zebrafish는 표준 방법 1에 따라 위치해와 reared 수 있습니다. 후반 애벌레 기간 과거 zebrafish의 발달 단계는 꼬리 지느러미 (posteriormost hypural 판)의 기지로 윗입술의 anteriormost 시점에서 자사의 표준 길이 (SL) 또는 밀리미터에서 직선 거리로 측정할 수 2. 약 개월 게시물 수정 (10-12밀리미터 SL)에서, 비강과 상악 동의 barbels는 각각의 후각 구덩이 근처와 maxillae의 뒷부분 모서리 등 투명 상피 봉오리를 나타납니다. zebrafish가 성숙으로, barbels가 좁은, 수염 같은 부속으로 연장. 상악 바벌가 큰 (2-3 ㎜)과 조작하기 쉬운이기 때문에, 우리의 프로토콜에만이 특정 부위요 적용, 유사한 기술, 그러나, 작은 코와 수염 적응 수 있습니다. 바벌 클리핑 <ol><li> 시스템 물에 산도 7.0에 버퍼 (에틸 3 aminobenzoate methanesulfonate) 0.015 % MS - 222에 침수하여 원하는 단계 (예., 1.5-2.5 cm SL ~ 3~6개월 포스트 - 수정)에서 성인 zebrafish을 마취시키다. 수영 동작 그만 때까지 관찰하고 길 환기가 느린 일반됩니다. 물고기의 크기에 따라 완료 마취 약 2~5분 걸립니다. </li><li> 수족관 엄마랑 사용하여 배양 접시에있는 서부 유럽 표준시 종이 타월에 접혀 조각 한 번에 1-3 물고기를 전송합니다. 무딘 주걱을 사용하여, 동양 그들이 서로 왼쪽 측면쪽으로 평행하게되도록 물고기. 절차를 수행하는 동안 피부와 아가미가 촉촉한 유지까지 operculum으로 몸 전체를 덮고 물고기 (길도 포함)의 꼬리 부분에 젖은 종이 타월을 접어 부분입니다. </li><li> 머리 지역을 밝히는 낮은 앵글 입사 광을 사용하여 stereomicroscope 아래의 페트리 접시를 볼 수 있습니다. maxilla의 사후 복부 코너 (그림 1)에서 protrudes 왼쪽 턱의 수염의 기본에 중점을두고 있습니다. <img src="/files/ftp_upload/1558/1558fig1.jpg" alt="그림 1" /> 그림 1. </li><li> 단단히 maxilla의 가장자리에 약간 말초 바벌의 샤프트를 파악. 바벌 샤프트를 강화하고 포셉 단 근위 미세 밀고 스프링 가위의 문턱을 삽입합니다. 가위는 안정 포셉에 감동하실 수 있습니다. 최첨단의 maxilla의 가장자리에 도달 때까지 바벌의 축을 따라 가위의 문턱을 슬라이드. 바벌 샤프트 잘리는 가위를 닫습니다. 절단 바벌, 아직까지 포셉에 사로잡혔다는 고정 또는 추가 관찰을 위해 제거할 수 있습니다. </li><li> 즉시 표면 곰팡이 감염을 제어하는​​ 파란색 메틸렌 한 방울을 포함하는 청소 시스템 물 (~ 500 ML)의 작은 수조에 물고기를 전송합니다. 물고기가 하루 복구할 수 있습니다. 다음날 아침, 양육 시스템에 물고기를 반환합니다. 바벌 재생 2 주 후에 최대한입니다. </li></ol> 일치하는 바벌의 쌍을 아가르 퍼가기 <ol><li> 적절한 안락사 기술로 물고기를 모아 4 ° C에서 밤새 교반과 호수 (PBS)를 버퍼 4퍼센트 50 ML 튜브 paraformaldehyde - 인산염의 수정. 정착액 볼륨은 조직의 최소 10 배 볼륨해야합니다. 고정 후, 승인된 용기에 paraformaldehyde 폐기물의 처분과 PBS의 여러 변화에 철저하게 조직을 씻어. </li><li> 2 % 250 ML 유리 플라스크 150 ML에서 준비 아가로 오스 (T M ~ 37 ° C) 증류수 인치 완전히 녹아까지 전자 레인지 또는 뜨거운 접시와 열, 주기적으로 agitating. 50-60 ° C의 물을 욕조에 녹은 솔루션을 평형. </li><li> 단단히 버퍼 챔버의 측면에 대해, 작은 gasketed 젤 곰팡이 (~ 100 ML 젤 볼륨 10 × 10cm) 배치하여 표준 젤 전기 영동 장비를 조립. 2-4 작은 이빨 샘플 빗 (4 X 1.5 mm)을 추가합니다. </li><li> 수준의 표면에 아주 얕은 우물을 형성 빗의 기반을 커버 그냥 금형에 녹아 아가로 오스를 부어. 즉시 온수 욕조에 다시 남은 아가로 오스를 넣어, 이것은 조직을 (9-12 단계) 포함 나중에 사용됩니다. 아가로 오스와 같은 온도로 피펫을 유지 술병에 긴 유리 파스퇴르 피펫 놔. </li><li> 20-30분에 대한 강화로 젤을 허용합니다. 빗를 제거한 후 버퍼 챔버에서 강화된 젤이 들어있는 겔 곰팡이를 제거합니다. 겔가 슬라이드되지 않도록 단단히 실험실 테이프로 겔 금형의 열린 측면을 테이프. 추가 아가로 오스가 (단계 13) 나중에 추가될 수 있도록이 테이프는 아가로 오스의 표면 여러 mm를 확장해야합니다. </li><li> stereomicroscope의 무대에 겔 금형을 놓습니다. 빈 우물을 확대하고 초점에 가장자리를 가져. </li><li> 잘 인접 아가로 오스의 표면에, 물 또는 고정 바벌의 표본 (예, 재생 및 제어)가 포함된 버퍼의 드롭을 피펫. UsiNG는 잘으로 촉촉한 표본을 드래그하여 아래로 밀어, 곤충 핀을 구부 러. 동양 바벌 샤프트의 서로 평행하고 잘의 반대 긴 가장자리에 대한 정렬. 표면 장력 위치에서 조직을 보유 도움이 될 것입니다. </li><li> 언제 포함 준비, 훌륭한 피펫 팁 또는 우물에서 여분 액체를 제거하는 실험실 조직의 모서리를 사용합니다. 조직가 건조하게하지 빨리 이렇게 작동합니다. </li><li> 예열 유리 파스퇴르 피펫을 사용하여 부드럽게 장소에서 바벌 조직을 봉인하기 위해 표본과 주변의 신선한 녹은 아가로 오스를 피펫. 너무 많이 넣다하지 마십시오. 아가로 오스는 견고하게하기 전에, 곤충 핀 지난 분을 조정합니다. </li><li> 잘 옆의 숫자 종이 표본 레이블을 장소와 아가로 오스의 작은 방울로를 확보. </li><li> 반복 표본의 각 잘 10을 통해 6 단계를 반복합니다. </li><li> 이미지 보정을위한 원하는 경우, 인쇄 마이크로 미터 규모 (예, 함께 종이를 삽입 <a href="http://incompetech.com/graphpaper/multiwidth/">http://incompetech.com/graphpaper/multiwidth/</a> 잘 빈의 맨 아래에). 우물의 바닥에 종이를 평평하게하고 따뜻한 아가로 오스로 커버. </li><li> 표본 모두 포함된 후, 젤의 표면은 아가로 오스의 경화 방울과 함께 불규칙한 것입니다. 평평한 표면에 젤을 놓고 상부 표면이 균일 때까지 부드럽게 녹아 추가 아가로 오스에 부어. 부드러운 표면을 얻기 위해 필요한 아가로 오스의 가장 작은 양을 사용하고 너무 아가로 오스는 전송 - 가벼운 사진을 흐릿하게 것입니다. 하든이 상위 계층을 허용합니다. </li><li> 젤 이제 각 바벌 쌍을위한 총 형태를 기록 stereomicroscope의 무대 사진 수 있습니다. 이미지 보정은 임베디드 마이크로 미터 규모를 사진 촬영하여 얻을 수 있습니다. </li><li> 전체 젤은 서부 유럽 표준시 종이 타올에 싸여 몇 주 동안 4 명이 한 비닐 봉투에 봉인 저장할 수 있습니다. </li><li> 자세한 분석을 위해, barbels의 일치하는 쌍을 포함하는 한천 블록은 메스 또는 레이저 블레이드로 젤 도려낸 수 있으며, 파라핀의 조직학에 대한 처리 또는 표준 방법에 의해 3,4 cryosectioning. 또는 개인 barbels는 물에 씻어서 잘 바늘과 아가로 오스 밖 해부 및 기타 다운 스트림 응용 프로그램 (예 : 전체 마운트 immunohistochemistry 또는 공촛점 현미경)을 처리할 수 있습니다. </li></ol>

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1+General+Methods+for+Zebrafish+Care.">Chapter 1 General Methods for Zebrafish Care.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Howe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+length:+Not+quite+so+standard.">Standard length: Not quite so standard.</a> Fisheries Research (Amsterdam). 56, 1-7 (2002).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+Embedding+Tissue+Samples+for+Sectioning.">Paraffin Embedding Tissue Samples for Sectioning.</a> Cold Spring Harbor Protocols. 6, (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+8.3+Agar+Embedding+for+Cryostat+Sectioning+of+Embryos+or+Larvae.">Ch 8.3 Agar Embedding for Cryostat Sectioning of Embryos or Larvae.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Hansen, A., Reutter, K., Zeiske, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+development+in+the+zebrafish,+Danio+rerio.">Taste bud development in the zebrafish, Danio rerio.</a> Dev Dyn. 223, 483-496 (2002).</li></ol>

zebrafish 턱의 수염은 zebrafish 여​​러 세포 유형의 성장, 유지 보수 및 재생을 공부 underutilized 조직 시스템입니다. 바벌의 돌출부가 더 인간 아날로그가 없다지만, 그것이 포함하고있는 세포 종류 고도 그것이 가능한 광학 명확하고 해부학적인 몸의 구조도 단순 원통형 구조로 피부 땀샘, melanocytes, 순환기 혈관과 신경을 연구하고, 보존하고 있습니다. 잘 공부 꼬리 지느러미와 마찬가지로, 바벌 조직이 절단하여 재생을 유도하실 수 있습니다. 해부 학적 랜드마크로 maxilla의 경계를 사용하여 절단 평면은 바벌 regrowth의 측정을 용이하게, 정확하게 삽입할 수 있습니다. 경험 교환 원을 연결 하시려면, 각각의 수술은 단 몇 초 정도 걸립니다. 복구는 신속하고, 우리는 지금까지 물고기의 행동에 아무런 단기 또는 장기 영향을 감지 없습니다. 한 상악 바벌 수영과 Zebrafish는 최대 6 개월 수술 후, 식사 및 비 수술 컨트롤처럼 효과적으로 번식하고, 조직 컬렉션의 시간을 비교 생존 있습니다. 이 수술의 생리적 영향 1) 바벌에서 실시 extraoral 입맛도 입술, 뺨과 머리 5 포함한 생선 상피의 다른 많은 부분에서 찾을 수 있기 때문에 최소한의 것으로 예측하고, 2) 차별 입맛이에 나타납니다 72 시간 내에 재생 바벌은 (레클레어 외., 게시되지 않은 데이터가 없습니다.) 이것은 상악 바벌 성인 척추의 컨텍스트 내에서 상처 치유, revascularization, 그리고 reinnervation 공부를위한 법으로서 시스템을 만듭니다. 중생의 수술 유도 후, barbels는 고정 조직의 morphometric 측정 및 / 또는 미세한 분석을위한 간격에서 수집된하실 수 있습니다. 편리, 상악 바벌는 cryosectioning 전체 마운트 immunohistochemistry, 그리고 현장에서 파라핀의 조직학 등 많은 표준 프로토콜의 응용 프로그램을 촉진 약 길이 (2-3 ㎜)와 zebrafish 배아의 직경 (1백~2백밀리미터)입니다 하이브 리다이 제이션. 함께 촬영이 기능은 턱뼈 ​​바벌 조직 수리 및 재생을 공부에 대한 생체내 모델에서 매우 가능합니다.

현미경 장비 전송 및 사고 광섬유 조명과 입체 해부 현미경은 바벌 클리핑과 한천 포함 필요합니다. 바벌 클리핑 <ul><li> 생선 시스템 물 </li><li> 이 zebrafish 크로싱 탱크 </li><li> MS - 222 (에틸 -3 - aminobenzoate methanesulfonate, 시그마 화학 # E10521 - 10G) </li><li> 무균 120 mm 페트리 접시 </li><li> 서부 유럽 표준시 종이 타올 </li><li> 뭉툭한 금속 주걱 </li><li> 더몬트 # 55 바이오 Inox 집게 (파인 과학 도구 # 11255-20) </li><li> 미니 Vannas 스프링 가위 (파인 과학 도구 # 15000-00) </li><li> 메틸렌 블루 antifungal 에이전트 (Drs. 포스터 &amp; 스미스 # CD - 905781) </li><li> 작은 수족관 엄마랑 </li></ul> 조직 수집과 한천 삽입 <ul><li> 1X의 4 % paraformaldehyde 인산염 버퍼 호수 (PF - PBS), 50 ML 원뿔 튜브에 aliquoted 및 -20 ° C에서 냉동 저장까지 사용 </li><li> 1X 인산염 버퍼 호수 (산도 7.27-6) </li><li> 250 ML 유리 술병 </li><li> 2퍼센트 증류수의 아가로 오스 </li><li> 표준 작은 아가로 오스 겔 전기 영동의 장비 (겔 크기 = ~ 10cm 정사각형) </li><li> 작은 이빨 전기 샘플 빗 (빗 사이즈 4 X 1.5 mm) </li><li> 온수 욕조 (50-60 ° C) </li><li> 실험실 테이프 </li><li> 블랙은 곤충 핀, 크기 000 (파인 과학 도구 # 26000-25) 에나멜 </li><li> 1-2 핀 홀더 (파인 과학 도구 # 26016-12) </li><li> 9 인치 유리 파스퇴르 피펫 </li><li> 피펫 벌브 또는 펌프 </li><li> 실험실 조직 </li><li> 작은 종이 라벨 </li><li> (옵션) 인쇄 교정 척도 </li><li> 서부 유럽 표준시 종이 타올 </li><li> 지퍼 - 폐쇄 비닐 봉투 </li><li> 메스 / 면도기 블레이드 (한천 블록을 잘라) </li></ul>

methods for the study of the zebrafish maxillary barbel

Barbels는 어류, 파충류와 양서류에서 발견 피부 감각 부속됩니다. zebrafish, Danio rerio는 barbels - 짧은 코에 한쌍과 긴 상악 쌍 이쌍을 개발하고 있습니다. 바벌 조직은 피부 세포, 땀샘, 입맛, melanocytes, 순환기 혈관과 감각 신경을 포함한 ectodermal, mesodermal 및 신경 크레스트 원산지 세포를 포함하고 있습니다. 대부분의 성인 조직과는 달리, 턱의 수염은 우리가 수명주기 전반에 걸쳐 이러한 조직 유형의 개발 및 유지 보수를 시각화 수 있도록 광학 분명하다. 이 동영상은 상악 바벌 (약 일개월 후 수정을 시작)의 조기 개발을 보여줍니다 및 성인 돌출부 (&gt; 삼개월 후 수정)의 재생을 유도하는 외과 프로토콜을 보여줍니다. 간단히, anesthetized 물고기의 왼쪽 상악의 수염은 maxilla의 꼬리 가장자리에 단지 말초 살균 포셉로 고가이다. 좋아, 멸균 스프링 가위는 절단 평면에 대한 해부 랜드마크를 설립,이 수준에서 바벌 샤프트를 잘라 포셉에 대한 위치입니다. 재생 성장하고, contralateral 바벌에 비해이 비행기와 관련하여 측정할 수 있습니다. 바벌 조직이 부상 2 주 이내에 최대한의 regrowth에 도달, 빠르게 다시 생성합니다. 재생 바벌을 분석하는 기술은 표준 DNA 전기 영동 젤의 우물에서 barbels의 일치하는 쌍 (재생 및 컨트롤)를 해부하고 삽입 등이 있습니다. 임베디드 표본이 편리 총 형태학 및 morphometry위한 stereomicroscope 아래 사진되며, 사전 등 파라핀의 조직학으로 하류 애플 리케이션에 몇 주 동안 저장 cryosectioning 및 / 또는 전체 탑재 immunohistochemistry 수 있습니다. zebrafish의 유전자 컨텍스트 내에서 다중 셀 유형의 재생 능력을 학습에 대한 생체내 조직 시스템의 소설로 상악 바벌 수립 이러한 방법.

Watch this Scientific Journal Video about Methods for the Study of the Zebrafish Maxillary Barbel at JoVE.com

Methods for the Study of the Zebrafish Maxillary Barbel

zebrafish 턱의 수염은 ectodermal, mesodermal 및 신경 크레스트 파생 상품을 포함 외피의 감각 기관이다. 중요한 것은 어른 바벌은 근위 절단 후 재생하실 수 있습니다. 이 동영상은 상악 바벌 개발을 소개하고 바벌의 표본의 수집, 퍼가기 및 다운 스트림 이미지 다음 재생을 유도하는 외과 프로토콜을 보여줍니다.

Zebrafish 상악 바벌의 연구 방법

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

methods-for-the-study-of-the-zebrafish-maxillary-barbel

Research

JoVE Journal

Biology

12.4K 조회수.  DePaul University. zebrafish 턱의 수염은 ectodermal, mesodermal 및 신경 크레스트 파생 상품을 포함 외피의 감각 기관이다. 중요한 것은 어른 바벌은 근위 절단 후 재생하실 수 있습니다. 이 동영상은 상악 바벌 개발을 소개하고 바벌의 표본의 수집, 퍼가기 및 다운 스트림 이미지 다음 재생을 유도하는 외과 프로토콜을 보여줍니다.

비디오: Methods for the Study of the Zebrafish Maxillary Barbel

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

zebrafish의 시각 기능을 연구하기 위해 optokinetic 응답을 사용하여

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

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, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Zebrafish의 Pronephros의 레이저 절제는 신장 상피 조직이 재생된를 공부

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

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 ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

Whitefly에서 Endosymbionts의 추출을위한 방법<em&gt; 베미 시아 타바시</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...