The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

동물에서 수행 모든 실험은 CICUAL 프로토콜 번호로, 실험 동물 관리 및 멕시코의 자치 대학교 (UNAM)의 사용을위한위원회의 승인을했다 : FLC40-14. (CICUAL &quot;Comité Institucional 파라 엘 Cuidado y를 USO 드 로스 짐승 같은 드하기 Laboratorio 델 연구소의 드 Fisiologìa Celular, 대학교 나시 오날 자치시 드 멕시코&quot;). 배아 단백질 추출물 1. 준비 <ol><li> 각 조건은 예를 들어 72 시간 포스트 수정 (HPF)를 위해, 원하는 단계에서 (morphants 대 야생형)을 비교하기 위해 200 배아 - (100)를 수집합니다. </li><li> 물고기의 물을 함유하는 배양 접시에 장소 배아는 (표 1 참조) 수동 뾰족한 집게를 사용하여 배아를 dechorionate. 이 대상 수용체을 소화 수 있으므로이 단계 (또는 프로토콜을 통해 다른 단백질 분해 효소) 동안 프로 나제​​를 사용하지 마십시오. </li><li> 1.5 ml의 미세 원심 분리 튜브에 넣어 배아 및 배아 TW 씻어1X 인산 완충 용액 (PBS)와 얼음 (표 1 참조). </li><li> deyolk 버퍼 500 μL를 추가 (표 1 참조). </li><li> (- 40 배 30) 용액에 배아를 부드럽게 아래로 피펫 팅에 의해 노른자의 대부분을 놓습니다. 72 HPF, 48 HPF의 배아를 들어 각각 파란색과 노란색 피펫 팁을 사용합니다. 노란색 끝은 약간 배아를 파괴하지 않도록 절단해야 할 수도 있습니다. 성공적인 노른자 자료는 현미경으로 배아를 관찰하여 확인할 수 있습니다. </li><li> 15 초 동안 600 XG에서 원심 분리하여 배아를 수집합니다. </li><li> 피펫을 사용하여 조심스럽게 상층 액을 버린다. </li><li> 워시 배아 회 500 ㎕의 세척 완충액으로 부드럽게 최저 속도 텍싱하여 (표 1 참조). </li><li> 15 초 동안 600 XG에서 원심 분리하여 배아를 수집합니다. </li><li> 여기에서 시작, 4 ° C에서 절차를 계속합니다. </li><li> 피펫을 사용하여 조심스럽게 상층 액을 버린다. </li><li> 용해 완충액 350 μL에 재현 탁 배아 (표 1 참조)과 플라스틱 유봉을 사용하여 균질화한다. </li><li> 4 ° C에서 테스트 튜브 로커에 용해 배아를 교반하면서 30 분을 품어. </li><li> 원심 분리하여 불용성 잔사를 제거하기 위해 15 분 동안 11,000 XG에서 배아를 용해. </li><li> 전송 신선한 튜브에 피펫을 이용하여 상층 액을 깨끗이 밀어 냈습니다. </li><li> 브래드 포드 (Bradford) 단백질 분석 (4), 또는 다른 적절한 방법에 의해 전체 단백질을 결정한다. 브래드 포드 분석법이 사용되는 경우가 4 표준 단백질의 과소 원인으로, 용해 버퍼에 세제 선물 동등한 농도의 존재 검량선을 수행한다. </li></ol> 2. 감지 내인성 수용체 단백질의 <ol><li> 선호도 라벨링 및 면역 침전 (모든 단계 4 O를 C에서) <ol><li> 1.5 ml의 미세 원심 분리 튜브에 총 배아 단백질의 500 μg의 버퍼 1 (표 1 참조) 1 μg의 / μL로 희석 - (400)를 배치합니다. 주 : 약 100 배의 총 단백질 500 μg의 획득하기 위해서 - 200 배아 초기 처리되어야한다. 72 HPF 배아는 정기적으로 betaglycan 검출을위한 충분한 총 배아 단백질의 ~ 5 μg의를 얻을. </li><li> 150 오후 최종 농도에 도달하고 4 ° C에서 테스트 튜브 로커 2 시간에 교반 배양 표시 TGF-β2 주식의 충분한 볼륨을 추가합니다. AFLIP이 시작되기 전에 Cheifetz 등. (5)에 의해 설명 된 바와 같이 TGF-β2는 클로라민 T 법에 의해 125 I로 표시해야합니다. 주의 : 내가 리간드 레이블 (125)를 처리하는 동안 노출을 최소화하기 위해 차폐. </li><li> (100) 희석 (배양이 O / N 수있다) 4 ° C에서 또 다른 2 시간 동안 배양을 계속 : 1에 도달하기 위해 관심있는 수용체에 대한 희석 항체의 충분한 볼륨을 추가합니다. 따라서 본 연구에서 사용하고 다른 3을 설명하지만,의 품질에 따라, 항혈청 번호 (31)의 최적의 희석이고tibody, 큰 또는 작은 희석 최적 일 수있다. </li><li> 적절한 면역 글로불린 결합 구슬의 50 μl를 추가합니다 (예를 들어, 이전에 TNTE에서 평형 1 재현 탁 G 단백질 세 파로스, : TNTE에서 원래 볼륨의 5)를 4 °에서 테스트 튜브 로커에 교반과 함께 50 분 동안 품어 기음. </li><li> 20 초 동안 11,000 XG에서 microcentrifugation으로 구슬을 복구 할 수 있습니다. </li><li> 적절한 방사성 쓰레기 용기에 상층 액을 버린다. </li><li> 20 초 때마다 11,000 XG에, 텍싱 및 microcentrifugation에 의해 IP 세척 버퍼 (표 1 참조) 1 ㎖로 IP-구슬 세 번 씻으십시오. </li><li> 재현 탁 IP-구슬 IP 세척 버퍼 250 μL있다. </li><li> (10 ㎎ / ㎖에서 DMSO에 용해 DSS) 숙신 이미 딜 수베의 1.5 μl를 추가합니다. 바로이 단계에서의 사용 전에 DSS 솔루션을 준비합니다. </li><li> 교반 4 ° C에서 15 분 동안 품어. </li><li> 가교 반응을 종결하기 위해, 500 ㎕의 O를 추가충분히 트리스 - CL pH 7.4의 재고 보충 F IP 세척 버퍼는 25 mM 트리스 - (CL)에 도달한다. 트리스의 유리 아미노산 그룹은 미 반응 DSS를 캡처합니다. </li><li> 20 초 동안 11,000 XG에 원심 분리기는 IP-구슬을 수집하고 뜨는을 폐기합니다. </li><li> 재현 탁 IP 비드 램리 완충액 30 μL 감소한다. </li><li> 삶아 샘플 94 ° C에서 5 분. </li><li> 선택적으로, 제조업 자의 프로토콜을 사용하여 감마 계수기에서 샘플을 분석한다. </li></ol></li><li> 샘플 분석 <ol><li> SDS-PAGE를 변성에 따라 샘플. 수용체의 질량에 해당하는 비율로 폴리 아크릴 아미드를 사용하여 표준 절차에 따라 젤을 실행합니다. </li><li> 고정 제 용액에서 겔을 담그고 실온에서 30 분 동안 (표 1 참조). </li><li> 15 분 동안 증류수로 젤을 씻으십시오. </li><li> 이전에 수화 필터 종이에 젤을 놓고 사란 랩 필름 커버. </li><li> 1 시간 동안 80 ° C에서 건조 젤. </li><li> 흰색은 phosphorimager의 scre에 젤을 노출RT O / N에서 엉. </li><li> 제조 업체의 프로토콜을 사용하여은 phosphorimager를 사용하여 노출 된 화면을 검색합니다. </li></ol></li></ol>

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The authors have nothing to disclose.

관심의 단백질에 대한 특이 항체와 서양 말의 사용은 배아 발생 동안 발현 7을 연구하는 유용한 도구입니다. 그러나, 높은 당화 단백질의 면역 인해 자신의 비효율적 인 전송 및 니트로 셀룰로오스 또는 PVDF 막 8,9 약한 결합을 매우 성공적으로되지 않았습니다. 프로테오글리칸 때문에 음전하를하고 폴리스티렌 표면 또는 소수성 블로 팅 막 중 하나에 잘 결합하지 않는 자신의 공유 부착 글리코 사 미노 글리 칸 체인 (GAG)으로, 이러한 단점의 좋은 예이다. 또한, GAG 사슬의 크기 이질성 효과적으로 오의 면적당의 양을 감소 겔의 섹터에 걸쳐 단백질을 &quot;확산&quot;. 관심의 단백질 발현 수준이 낮은 경우뿐만 아니라, 정기적 인 웨스턴 블랏에 의해 그 검출은 어려운 작업이다. 수용체 (TGFBR3) 또는 betaglycan β 유형 III 형질 전환 성장 인자(BG), 제브라 피쉬 3 개의 GAG의 부착 포유 동물 1 사이트와 하나 막 수용체는 이러한 특성을 모두 가지고있다. 이러한 장애물을 극복하는 프로토콜이 고안되었습니다 만, 아비딘 - 비오틴 복합체 (ABC) 시스템 10, 11 또는 양으로 대전 된 막 (12)에 프로테오글리칸의 immunobloting에 의해 검출처럼, 그들은 같은 프로토콜에서 두 문제를 해결할 수 없습니다. 자연 리간드 TGFβ2 대한 특정 항체의 가용성 BG의 높은 친 화성을 활용 AFLIP, 친화 라벨링 및 면역 침전의 이점을 결합하는 기술들은, 위에서 웨스턴 블롯 검출 한계는 극복 될 수 논의. 자신의 방사성 표지 된 리간드와 막 결합 수용체의 선호도 표시는 몇 가지 중요한 성장의 식별 및 특성에 대한 쓸모 배양 된 세포 13-16에서 수용체 요인했습니다 잘 사용하는 도구입니다.세포 단층을 라벨링 종래 친 화성 조직 배양 접시에 리간드 라벨 ​​공유 결합을 실시하고 용해되지만, AFLIP에서 리간드 - 수용체 복합체는 제 배아 용해질 다음, 면역 침전에 의해 정제하여 최종적으로 공유 가교 결합을 형성한다. 왜냐하면 방사성 리간드의 사용으로, AFLIP 다른 성장 인자 또는 표지 된 리간드와 함께 좋은 항체를 사용할 수있는 사이토 카인 수용체에 적용 할 수있는 매우 중요한 원칙이다. AFLIP이 가능성이 낮은 풍부하지만 생리 관련 분자의 검출을 도움이 될 것이다. 이 때문에 친 화성 라벨의 가교 공정에서의 고유의 변동, AFLIP 정량적 측정 수용체​​의 수준을 결정하기 위해 사용될 수 없다. 적절한 제어 병렬로 수행하는 경우에는, 그들의 발현의 상대 수준의 매우 정확한 계측을 제공 할 수있다. AFLIP 총 단백질 추출물을 사용하여, 그것은 restri되지해부 성인 표본에 적용하는 경우, 수용체의 하나의 특정한 셀룰러 위치 cted, 그것은 전체 개인 또는 기관에서의 발현을 보여준다. 필요한 AFLIP 다른 생화학 프로토콜에 링크 될 수 있다면 마지막 탄수화물 효소 digestions 같이, 상기 관심의 수용체를 특성화한다. 기존의 친 화성 라벨링과 유사하게, 갓 (125) 내가 표지 리간드는 것이 좋습니다. 125 I의 짧은 반감기를 고려할 때, 리간드 신선한 동위 원소로 라벨링 후 처음 2 주 이내에 사용해야합니다. 프로토콜의주의 사항의 대부분이 해당 단계에서 언급되었지만, 하나의 특별한 언급은 가능한 한 완전하고 균일해야 노른자의주의 제거에 제공해야한다. 노른자의 불균일 한 금액을두면 해물에서 선의의 배아 단백질의 잘못된 정량 될 것입니다.

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

<table border="0" cellpadding="0" cellspacing="0" width="670">
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			<td height="40" style="height:40px;width:216px;">Disuccinimidyl suberate (DSS)</td>
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			<td height="40" style="height:40px;width:216px;">Typhoon 9400</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">63-0055-78</td>
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			<td height="20" style="height:20px;width:216px;">Cobra II Auto gamma counter</td>
			<td style="width:168px;">Packard</td>
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			<td height="20" style="height:20px;width:216px;">Exposure Cassette</td>
			<td style="width:168px;">Molecular Dynamics</td>
			<td style="width:119px;">63-0035-44</td>
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			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3624</td>
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			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3040</td>
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			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3828</td>
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			<td height="21" style="height:21px;width:216px;">K2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3246</td>
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			<td height="21" style="height:21px;width:216px;">CH4O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9070</td>
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			<td height="21" style="height:21px;width:216px;">C2H4O2</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9508</td>
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			<td height="21" style="height:21px;width:216px;">CH2O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">2106</td>
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			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">L4509</td>
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			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:168px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3306</td>
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			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
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			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
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			<td height="63" style="height:63px;width:216px;">Crystal Sea Marine Mix</td>
			<td style="width:168px;">Marine Enterprises International</td>
			<td style="width:119px;">http://www.meisalt.com/Crystal-Sea-Marinemix</td>
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</table>

affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

그림 1은 AFLIP 얻은 대표적인 결과를 보여줍니다. 125 I-리간드에서 온 레인 1 신호는 공유 (150 kDa의 마커 아래 BG 코어) 제브라 피쉬 betaglycan 핵심 단백질 또는 글리코 사 미노 글리 칸의 첨부 파일 (GAG, 도말하여 프로테오글리칸 형태로 처리 된 BG 코어 중 하나에 연결된 ) 170 kDa 내지 겔의 상단까지. 마이그레이션이 패턴 날카로운 코어 단백질 플러스 (GAG 때문에 사슬의 길이 이질성)를 도말 프로테오글리칸은 TGFBR3이 특징이다. DSS에 공유 결합 매 리간드 - 수용체 복합체 형성에 연결되지 않기 때문에, 겔 (125 I-TGF-β2)의 이동 앞에 나타나는 125 I-리간드 무연있다. 이 유리 리간드가 수용체에 결합하고, 결합 된 면역 중에 남아 있지만 공유 결합 부착되지 때문에, SDS-PAGE 과정에서 분리. 아니netheless으로, 그 신호의 세기가 여전히 표지 수용체에 의해 주어진 하나로 상관. 이것은 강도에 대응 GAG 및 BG 코어에서 약한 신호에 의해 알 수있는 72 HPF 배아 3보다 BG 낮은 양, 제브라 피쉬 배아 HPF (48)에서, 레인 2 및 3과 비교하여 나타내 알 수 겔의 전면에 나타나는 유리 리간드에 의해 주어진 신호 (레인 2). AFLIP 사용할 때 쥐에 대한 항체가 BG 6 ZF BG와 교차 반응하지 않는 점을 감안, 항체는 음성 대조군 (레인 3) 역할을, 따라서 아무런 신호도주지 않았다. 유사 부정적인 결과를 방지 ZF BG 항체 (3)의 전 면역 혈청을 얻었다. 도 2는 AFLIP에 의한 모르으로,이 경우, 실험 조작을 실시 배아 betaglycan의 감소는 주어진 수용체의 발현 수준을 측정하는 방법을 보여주는이 켜졌 3. 레인 2는 ZF BG 차 전 사체의 exon2-intron2 경계에 관한 모르 폴리 7 NG 투여의 효과를 나타내고있다 레인 (1)는, HPF (72) 야생형 배아 TGFBR3의 수준을 나타낸다. 이 모르 폴리 노 얻은 BG 다운 규제는 그 잘못 일치 제어 (레인 3)에 의해 재생되지 않습니다. 이 결과는 세 이전에 기재 한 바와 같이 얻은 모르 표현형이 TGFBR3의 최저 특정 것을 확인 하였다. <img alt="그림 1" src="/files/ftp_upload/54405/54405fig1.jpg" /> Zebrafish의 태아에 BG의 그림 1. AFLIP 탐지. 72 시간 포스트 수정 (HPF)에서 배아에서 총 단백질 추출물 (레인 1, 3) 48 HPF (레인 2) 다음에 125 I-TGF-β2 친 화성 라벨링을 실시 하였다 BG (레인 3 RBG 토끼 항 제브라 BG (레인 1 및 2 zBG) 또는 토끼 항 - 래트와 IP에 의해) 제어 등. 자가 방사선에 BG는 GAG (BG 코어)없이 검출 할 수 또는 글리코 사 미노 글리 칸 수정 BG (GAG)로. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54405/54405fig2.jpg" /> 그림 2. BG 모르 폴리 노 최저입니다. AFLIP에 의해 감지 된 특정 BG 모르 폴리 노 (레인 2) 또는 불일치 제어 (레인 3)과 미세 주입 치료 WT 배아 (lane1)에서 BG의 125 I-TGF-β2 친 화성 라벨. 만 (CPM) 당 카운트는 면역 및 가교 단계가 표시됩니다 후. 회복 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-wi 번째-next.within 페이지 = &quot;항상&quot;&gt; <tbody><tr><td> 완충기 </td><td> 구성 </td></tr><tr><td> 버퍼 1 </td><td> pH가 7.5의 50 mM 트리스 -HCl, 150 mM의 염화나트륨, 0.1 % 트리톤 X-100 </td></tr><tr><td> Deyolk 버퍼 </td><td> 55 mM의 염화나트륨, 1.8 밀리미터의 KCl, 1.25 밀리미터의 NaHCO3 </td></tr><tr><td> 물고기 물 </td><td> 자료 표를 참조하십시오 </td></tr><tr><td> 정착액 솔루션 </td><td> 50 % CH 3 OH, 12 % CH 3 COOH, 0.185 %의 HCHO </td></tr><tr><td> IP 세척 버퍼 </td><td> 10 mM의 나트륨 2 HPO 4, 2 mM의 KH 2 PO 4, 137 mM의 염화나트륨, 2.7 mM의 KCl을 0.1 % 트리톤 X-100, 0.02 % SDS, pH 7.4의 </td></tr><tr><td> 램 믈리 버퍼 </td><td> 2 % SDS, 10 % 글리세롤, 100 mM의 DTT, 60 mM 트리스 (산도 6.8), 0.001 %의 브로 모 페놀 블루. </td></tr><tr><td> 용해 완충액 </td><td> 50mM 트리스 - 염산 pH 7.4로 150 mM의 염화나트륨, 1 mM의 EDTA, 1mM의 PMSF, 0.5 % 트리톤 X-100, 0.1 % SDS, 0.5 % 나트륨 Deoxych올 레이트 </td></tr><tr><td> PBS </td><td> HPO 4 137 mM의 염화나트륨, 2.7 mM의 KCl을 10 mM의 나트륨 2, 2 mM의 KH 2 PO 4 pH 7.4로 </td></tr><tr><td> TNTE </td><td> 50mM 트리스 - 염산 pH 7.4로 150 mM의 염화나트륨, 1 mM의 EDTA, 0.1 % 트리톤 X-100 </td></tr><tr><td> 세척 버퍼 </td><td> 110 mM의 염화나트륨, 3.5 밀리미터의 KCl, 2.7 mM의 염화칼슘 2, 10 mM 트리스 - 염산 pH가 8.5 </td></tr></tbody></table> 표 1 : 버퍼 조성물

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Zebrafish의 배아에서 내인성 수용체의 친화력 레이블 검출

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.6K 조회수.  Universidad Nacional Autónoma de México. A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

비디오: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish의 태아에서 기계 선박 부상

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

연구

발생학

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

배아의 형광 라벨의 DNA 특정 얼룩 메틸 녹색의 응용 프로그램

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

분리 및 Zebrafish의 배아에서 단일 세포의 특성

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Blastulae 개발의 외과 융합에 의해 Parabiotic 제브라 피쉬 배아의 생성

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

열 충격이 치료와 제브라 피쉬 배아의 배수성 조작

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

살아있는 개구리와 Zebrafish 태아의 저산소증 유도

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Zebrafish 초기 배아에 종양 세포 전기 활동의 시각화

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

전체 얼룩말 배아의 연장된 시간 경과 공초점 현미경 검사법을 위한 레이어형 장착 방법

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

제브라피시 배아와 애벌레의 전체 마운트 면역항암화학

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...