Confocal 현미경에서는 미국 국립 과학 재단 (NSF), 부여 #DBI-0722569 자금으로 구입 했습니다. 연구로는 미국 국립 연구소의 건강/국립 연구소의 일반 의료 과학 (NIH/NIGMS) 그랜트 #GM085290 및 미국 부서 방어 (국방부) 부여 #W81XWH-16-1-0466 R.M. 브루 스 터에 게 수 여를 지원 했다. E. 생체 전 대학 및 학부 과학 교육 프로그램을 통해 하 워드 휴즈 의학 연구소에서 UMBC에 교부 금에 의해 지원 되었다, # 52008090를 부여. S.P. 브라운에 의해 미국 부의 교육 GAANN 장학금을 지원 했다, #GM055036, 그리고 자금으로 미국 국방부 부여 #W81XWH-16-1-0466 연구 조교 메이어 대학원 친교 NIH/NIGMS 보조금에 의해 자금.

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저자는 공개 없다.

현재 많은 방법이 있다 초기 zebrafish 개발에서 산 역학 이미징을 위한 조직11,12,,1314고정 태그 분자의 라이브 영상에서의 immunolabeling에 이르기까지. 단일 셀에 MTs 동적 또는 안정 상태에 있을 수 있습니다, 있지만 epithelialization는 MTs는 시간이 지남에 따라 점차적으로 안정화 과정 이다. 안정적이 고 동적...

Microtubules (MTs)는 중합체의 α-및 β-tubulin 선형 protofilaments, 몇 가지는 속이 빈 튜브1,2형태로 결합으로 조립 이다. MTs는 끝 플러스 빠른 성장 하 고 느린-성장 하 고 끝은 centrosome 또는 다른 microtubule 조직 센터 (MTOC)3에서 앵커는 마이너스와 대립 된 구조. 드 노 보 MT 형성 nucleation 복잡 한 γ-tubulin 반지에서 (γ-TURC), 마운트 어셈블리4에 대 한 서식 파일을 제공 하 여 시작 됩니다. 어떤 주어진된 셀에 MTs의 두 인구 구분할 수 그 차례 이상의 서로 다른 속도로. 동적 MTs 성장의 단계 동적 불안정5라고 하는 과정에서 수축으로 전환 하 여 그들의 세포질 환경 탐구. 동적 MTs와 달리 안정적인 MTs 비 성장 고 동적 MTs6보다는 더 긴 반감기.

세포 생물학에 있는 연구의 십 년간은 정교한 배열의 마운트 구조와 기능을 공부 하는 도구를 제공 하 고 이러한 cytoskeletal 요소에 지식의 큰 몸이 귀착되. 예를 들어, MTs 설립 및 유지 보수 뿐만 아니라 안정적인 동적 MTs7, 대의 차동 subcellular 배포에 뿐만 아니라 그들의 본질적인 극성에 기인은 세포 극성의 중심 역할을 담당할 8. 반면, 훨씬 덜은 이해 MT 아키텍처 및 척 추가 있는 태아 같은 더 복잡 한 3 차원 (3 차원) 환경에서 기능에 대 한 부분에서 높은 해상도에서 산 골격을 이미징의 도전 때문. 이 제한에도 불구 하 고 최근 세대 GFP 표현 유전자 변형 라인 라벨 MTs 또는 붙일 태그가 MT 마커의 과도 식 MTs 받 다 동적 변화 및 그들의 휴대에 대 한 우리의 이해를 증가 했다 고 zebrafish 태아에 있는 발달 역할. 전체 MT 네트워크 수 수 몇 군데는 tubulin에 유전자 변형 라인에 직접 레이블된9 또는 tubulin 고분자는 직접 표시 하지 MT 관련 된 단백질을 사용 하는 Doublecortin 같은 키 (Dclk) 또는 Ensconsin (EMTB)10, 11. 다른 선 (및 구문) 생성 된 산 플러스 끝 으로써 특히 산 내장 극성의 평가 활성화 또는 끝11,12,13, 마이너스 centrosome 고정 14. 이러한 도구의 힘 유기 체를 개발, MT의 역학을 공부 하는 능력에 있다. 이러한 연구 결과 밝혀졌다, 예를 들어 특정 세포 인구에서 MTs의 공간 및 동적 배포, mitotic의 방향 스핀 들 겪고 morphogenesis (세포 분열의 비행기의 표시기), 조직에 산 폴리머의 극성 세포 신장 등 마이그레이션 프로세스와 관련 및 MT 성장률 혜성 속도9,,1315에 의해 결정. 이러한 도구의 한계는 그들이 쉽게 안정적이 고 동적 MT 인구 사이 차별 하지 않습니다 이다.

풍부한 세포 생물학 문학에서 그리기, zebrafish 태아에 있는 안정적이 고 동적 MTs 이미지를 immunolabeling 방법 설명, 여기는 유전자 변형 라인의 사용을 보완 합니다. 제 브라에서 같은 immunolabeling 방법의 광범위 한 사용은 고정 절차 동안 MT 무결성을 보존 하기에 있는 어려움에 의해 약간 방해 되었습니다. 프로토콜 1 immunolabeling 총, 동적, 최적화 된 방법을 설명 하 고 있는 안정적인 MTs 개발 zebrafish hindbrain의 횡단면. 또한, 상업적으로 사용 하 여 간단한 메서드 사용 가능한 소프트웨어 계량 이러한 산 인구를 설명 되어 있습니다. 안정적인 MTs는 α-tubulin acetylation, detyrosination, 시간16,17동안 안정적인 MTs에 축적 등의 여러 포스트 번역 상 수정에 따라 동적 MTs에서 구별 된다. Zebrafish 태아 acetylation 속눈썹 및 axonal MTs만 안정적인 interphase MTs18, 안정된 MTs의 하위 집합을이 마커의 유용성을 제한 하지에 발생 합니다. 대조적으로, detyrosination18zebrafish 태아 있는 모든 안정적인 MTs에 나타납니다. 이 포스트 번역 상 수정 α-tubulin (detyrosinated tubulin)18 의 카 터미널 글루탐산을 노출 하 고 안티-Glu-tubulin19를 사용 하 여 검출 될 수 있다. Detyrosination 안정 MTs에 우선적으로 발생 합니다, 하지만 실험적인 증거가 포스트 번역 상 수정 산 안정성16의 원인 보다는의 결과 임을 나타냅니다. 동적 MTs 구성 상호 MT 인구, 항 체, 안티-Tyr-tubulin, 그 구체적으로 인식 하는 α-tubulin19의 tyrosinated 형태를 사용 하 여 구별 된다. 이러한 마커 및 confocal 영상 immunolabeling, 다음 MTs (길이, 번호, 및 관계 되는 풍부)의 정량 분석 개발 신경 튜브의 정의 된 영역에서 수행할 수 있습니다. 효율적인된 방안은 여기 3 차원 이미지 처리 소프트웨어를 사용 하 여이 분석을 수행 하기 위해 제공 됩니다. 이 메서드는 주소 morphogenesis 설립 또는 세포 극성20의 성숙에 관한 질문에 적용할 수 있습니다. 실제로, 안정적인 MTs의 편광 배열의 정교 포토 리셉터 morphogenesis21, 개발 신경 튜브18 및 축 삭 형성8셀의 epithelialization를 포함 하 여 많은 개발 이벤트를 동반 한다.

프로토콜 2 그들의 어셈블리 단계 (nucleation/앵커리지와 성장)22,23동안 MTs를 분석 하는 세포 생물학 분석 실험의 비보에 적응을 설명 합니다. 초기 MTs는 centrosome에서 nucleated 되며 이후 어머니 중심소체23의 subdistal 부속에 고정. Depolymerization를 다음 초기 MT 자라나 분석 하는 방법을 설명 합니다. 이 프로토콜 MTs, 약 희미하게 절차와 치료 후 회복 기간 depolymerize nocodazole 치료에 세부 정보를 제공 합니다. MT 다시 성장에서 정기적 모니터링총 MTs에 대 한 마커 immunolabeling에 의해 s 게시물 유실 (β-tubulin) 안티는 centrosome에 대 한 표시와 함께 (안티 γ-tubulin)과 핵 (4', 6-diamidino-2-phenylindole (DAPI)), 프로토콜 1에서 설명 하는 일반 절차에 따라. 이 프로토콜의 MT depolymerization 단계 드 노 보 산 성장에 대 한 평가 보다는 기존 MTs의 확장 하면 필수적 이다. 이 기술은 다른 측정 하 산 성장 속도 (depolymerization의 부재) 끝 트 란 외.와 같이 녹색 형광 단백질 (GFP EB3), 융합 단백질 3을 묶는 등 플러스 팁 마커를 사용 하 여 다른 게시 된 절차 이므로 201211. 또한,이 분석 결과 배아 드 노 보 마운트 어셈블리에 결함 분석에 특히 유용 이전에 보고 된 NEDD1 돌연변이는 γ-tubulin는 centrosome에의 채용은 장애인 등에서 불완전 한 결과 신경 관 형성 및 신경 결함24.

<table border="0" cellpadding="0" cellspacing="0" width="916">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="140">
			<td height="140" style="height:140px;width:207px;">Low Melting Point Agarose</td>
			<td style="width:177px;">IBI scientific</td>
			<td style="width:159px;">IB70050</td>
			<td style="width:373px;">Only used for embedding. 
			 
			Prepare 4% LMP agarose 
			by heating a solution of&#160; 4 grams LMP agarose per 100 ml 1X TBS in a microwave 
			until polymerized. Keep warm on a 50 &#730;C hot plate with the cap secured</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Agarose</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">Used to treat petridishes.&#160;&#160; Prepare 1% agarose 
			by heating a solution of&#160; 1 gram agarose per 100 ml 1X embryo medium in a microwave 
			until polymerized.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Nocodazole</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">487928-10MG</td>
			<td style="width:373px;">Make stock by dissolving entire bottle of powder in distilled water and aliquoting into 500ul aliquots.&#160; Store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">8% Formaldehyde</td>
			<td style="width:177px;">Electron Microscopy Sciences</td>
			<td style="width:159px;">157-8-100</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Kpipes</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P7643</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">NaCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">S7653</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Tris-HCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">T3253-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">KCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P9333-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">CaCl2&#183;2H2O</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">C5080</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">NP-40</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB01424</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">EGTA</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">E3889-25G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">MgCl2</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">M2670-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Normal Goat Serum</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">S26-100ml</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Bovine serum albumin (BSA)</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">BP1605</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Triton-x</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB02025</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Pronase E (non-specific Protease from Streptomyces griseus)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P5147-1G</td>
			<td style="width:373px;">make stock solution by diluting 10mg per ml of ddH2O. Aliquot in 1ml aliquots and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Anti-Fade mounting medium</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">P10144</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">DAPI</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">D1306</td>
			<td style="width:373px;">Prepare 50 ml DAPI solution by first combining 1 &#181;l DAPI stock&#160; with 1 ml sterile water and then adding the DAPI/water mix to 49 ml 1X TBS; store in foil at 4 &#730;C for months.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:207px;">Mouse anti-&#946;-tubulin</td>
			<td style="width:177px;">Developmental studies Hybridoma Bank</td>
			<td style="width:159px;">E7</td>
			<td style="width:373px;">1/200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#947;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX113286</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#945;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX108784</td>
			<td style="width:373px;">1/1000*</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-detyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">AB3201</td>
			<td style="width:373px;">1/200-1/1000*&#160; Titrate antibody with first use of new lot.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-tyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">ABT171</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Mouse anti-centrin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">04-1624</td>
			<td style="width:373px;">1/1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11008</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11012</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11005</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11001</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Vibratome</td>
			<td style="width:177px;">Vibratome</td>
			<td style="width:159px;">1500</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Forceps</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td style="width:159px;">555227F</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">100 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229693</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">35 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229638</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">50 ml falcon tube</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-432-22</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Woven nylon mesh 70 um</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">B0043D1SZG&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Micropipette</td>
			<td style="width:177px;">Gilson</td>
			<td style="width:159px;">F123602</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Glass pipette</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">NC-999363-9</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Aquarium sealant</td>
			<td style="width:177px;">Amazon.com, by MarineLand</td>
			<td style="width:159px;">Silicone Sealer 1 oz (Tube)&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Ring stand</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-675BO</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Microbore PTFE Tubing, 0.022&#34;ID</td>
			<td style="width:177px;">Cole-Parmer</td>
			<td style="width:159px;">WU-06417-21</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Modeling clay</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">Sargent Art 22-4000</td>
			<td style="width:373px;">Any wax or oil based non-toxic modeling clay will suffice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Clamp</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">02-215-466</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">60ml syringe</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-820-11</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:207px;">Embryo medium (E3)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">34.8 g NaCl 
			1.6 g KCl 
			5.8 g CaCl2&#183;2H2O 
			9.78 g MgCl2&#183;6H2O 
			 
			To prepare a 60X stock, dissolve the ingredients in H2O, to a final volume of 2 L. Adjust the pH to 7.2 with NaOH. Autoclave. To prepare 1X medium, dilute 16.5 mL of the 60X stock to 1 L.&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Blocking Solution</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">50 ml TBS-NP-40 
			2.5 ml normal goat serum 
			1 g BSA 
			625 &#181;l Triton-X</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">TBS-NP-40 (pH 7.6)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">155 mM NaCl 
			10 mM Tris HCl 
			0.1% NP-40</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">2x MAB (pH6.4)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">160 mM KPIPES 
			10 mM EGTA 
			2 mM MgCl2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Commercial 3-D Image processing Software</td>
			<td style="width:177px;">PerkinElmer</td>
			<td style="width:159px;">Volocity (V 6.2)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dry block heater&#160;</td>
			<td style="width:177px;">VWR</td>
			<td style="width:159px;">12621-108</td>
			<td style="width:373px;">Used as a hot plate to melt agarose in Protocol 1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dissecting Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">MZ12</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Confocal Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">SP5</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Flat embedding mold</td>
			<td style="width:177px;">emsdiasum.com</td>
			<td style="width:159px;">BEEM 70904-01</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Public domain image processing software</td>
			<td style="width:177px;">NIH</td>
			<td style="width:159px;">ImageJ (V 1.5)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:532px;">* Success varies by lot number</td>
		</tr>
	</tbody>
</table>

use of immunolabeling to analyze stable, dynamic, and nascent microtubules in the zebrafish embryo

Microtubules (MTs)는 역동적이 고 연약한 구조는 이미지에 vivo에서, 특히 척 추가 있는 태아에에서 게 도전 하는. Immunolabeling 메서드는 zebrafish 태아의 개발 신경 관에서 MTs의 명백한 인구 분석 여기 설명 되어 있습니다. 신경 조직에 포커스를 실행 하는 동안이 방법론은 다른 조직에 광범위 하 게 적용 됩니다. 그러나 절차는 일찍 중반 somitogenesis 단계 배아 (1 somite 12 somites에), 그들은 수 다른 단계 비교적 사소한 조정의 범위에 적응을 위해 최적화 됩니다. 첫 번째 프로토콜에는 안정적이 고 동적 MTs의 공간적 분포를 평가 하 고 이미지 처리 소프트웨어와 함께 이러한 인구의 정량 분석을 수행 하는 방법을 제공 합니다. 이 방법은 기존 도구 이미지 microtubule 역학 및 실시간, 사용 유전자 변형 라인 또는 태그 구문 과도 식에서 배포를 보완합니다. 그러나 사실, 이러한 도구는 매우 유용 합니다, 쉽게 역동적이 고 안정적인 MTs 사이 구분 하지 않습니다. 이미지 분석이 고유한 microtubule 인구를 셀 분극 및 morphogenesis 기본 이해 메커니즘에 대 한 중요 한 의미를 갖는다. 두 번째 프로토콜 초기 MTs를 구체적으로 분석 하는 기법을 설명 합니다. 이것은 약 nocodazole와 마약 유실 후 복구 기간 microtubule depolymerization 다음 시간이 지남에, MTs의 드 노 보 성장 특성을 포착 하 여 수행 됩니다. 이 기술은 zebrafish 태아에 있는 MTs의 연구에 적용 하지 않은 하지만 microtubule 어셈블리에 연루는 단백질의 기능 vivo에서 조사에 대 한 가치 있는 분석 결과 이다.

Immunolabeling를 사용 하 여 안정적이 고 동적 MTs의 분석 프로토콜 1, 산 초 (신경 용골) 동안 부분 모집단의 분포와 신경 관 발달의 늦은 (신경 대) 단계 드러났습니다, 각각 사용 하 여 Glu tubulin 및 Tyr tubulin 표식으로 안정적이 고 동적 MTs에 대 한. 동적 MTs는 hindbrain에 신경 용골 단계 (4-5 somites)에서 지배 (그림 2A-D). ...

Watch this Scientific Journal Video about Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo at JoVE.com

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Immunolabeling 방법 개발 zebrafish 두뇌에 microtubules의 명료한 인구를 분석 하는 다른 조직에 광범위 하 게 적용 되는 여기에, 설명 됩니다. 첫 번째 프로토콜 immunolabeling 안정적이 고 동적 microtubules에 대 한 최적화 된 방법을 설명합니다. 두 번째 프로토콜 이미지와 초기 microtubules 구체적으로 정량화 하는 방법을 제공 합니다.

분석, 동적, 안정과 초기 Microtubules Zebrafish 태아에 있는 Immunolabeling의 사용

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

use-immunolabeling-to-analyze-stable-dynamic-nascent-microtubules

Research

JoVE Journal

Developmental Biology

8.2K 조회수.  University of Maryland, Baltimore County. Immunolabeling 방법 개발 zebrafish 두뇌에 microtubules의 명료한 인구를 분석 하는 다른 조직에 광범위 하 게 적용 되는 여기에, 설명 됩니다. 첫 번째 프로토콜 immunolabeling 안정적이 고 동적 microtubules에 대 한 최적화 된 방법을 설명합니다. 두 번째 프로토콜 이미지와 초기 microtubules 구체적으로 정량화 하는 방법을 제공 합니다.

비디오: Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

생산 및 선택적으로 형질 도입 기본 희소 돌기 아교 세포 전구체 세포에 렌티 바이러스의 사용을위한<em&gt; 체외</em&gt; 수초 분석

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

연구

발생학

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

코스닥 시스템의 사용은 분자 메커니즘 Zebrafish의 재생의 연구를

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebrafish의 배아에 주입 마이크로 비드

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

살아있는 Zebrafish의 배아에서 세포 이하 구조를 이미징

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

라이브 Zebrafish의 배아에서 유사 분열 부문 및 역학을 관찰

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

척색에서 배아 미세 주입 및 Electroporation에<em&gt; Ciona intestinalis의</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM와 Microrheology Zebrafish 태아 노른자위 셀에

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

신경 세포에서 세포내 수송 분석 꼬마 선 충 의 동원 정지

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

두 Salmonid 물고기, 공기 스트립의 효과 (Oncorhynchus Mykiss) 무지개 송어와 브라운 송어 (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...