우리는 마우스 사육 및 관리와 지원 및 실험실 관리 K. 길 주셔서 감사합니다. 또한 기술적인 도움을 무코 야마 연구실 회원에게 감사드립니다. 자금은 보건 Naitonal 연구소의 교내 연구 프로그램에 의해 제공되었다.

1. 수집 마우스 배아 림브 스킨 (E13.5 ~ E17.5) <ol><li> 안락사는 승인 절차에 의해 암컷을 연결. 우리의 승인된 동물 프로토콜에 따르면, 여자는 CO 2 노출에 의해 euthanized하고 다음 경추 전위에 의해 안심하시기 바랍니다. 의 흡수 종이 타월에 동물을 배치하고 스퀴즈 병에서 70% EtOH / H 2 O에서 철저하게 그것을 만끽해보세요. </li><li> 자궁은 그대로 해부하다 피로를 씻어 차가운 행크스 &#39;밸런스드 소금 솔루션 (HBSS)이있는 100 X 15mm 페트리 접시에 놓으십시오. </li><li> 배아를 분리하고 해부하다. 배아에서 매우 얇은 양막을 제거합니다. </li><li> 각각의 배아는 genotyped해야하는 경우 (옵션) 35 X 10mm 페트리 접시에서 단일 배아를 해부하다. 해부 꼬리는 genotyping의 0.2 ML PCR 튜브로 전송됩니다. </li><li> 배아의 앞발로를 차단하고 전송 인산 버퍼 살린 (PBS)에 얼음처럼 차가운 신선한 4 % Paraformaldehyde (PFA) 2 ML을 포함한 24 잘 접시에 고리 집게로 앞발. </li><li> 4 ° C 하룻밤에 Nutator 믹서에 부드러운 믹싱으로 앞발을 고정. </li><li> 다음날, PFA를 제거하고 실온에서 Nutator 믹서에서 부드러운 혼합과 PBS 2 ML 5 분 세 번 씻으십시오. </li><li> 100 % 메탄올 (MeOH)에 앞발을 전송하고 -20 ° C의 효소 냉동고 (임계 온도 제어와 자동 서리를 없애다 기능없이 냉동실)에 그들을 저장할 수 있습니다. 차 항체는 100 % MeOH 치료 후 표 1에 나열된 작업. </li><li> 고급 핀셋을 사용하여 forelimb에서 출발 피부 껍질. 림브 피부, 탈수, 갈기갈기 쉽게 분리해야합니다. 우선 직면 복부 측면 (손바닥 쪽)와 다리를 놓으십시오. 동영상과 같이 다음 고급 핀셋을 사용하여 피부를 잘라. 다음 손상없이 부드럽게 피부를 벗겨내는, 전체를 사지 주변 해부하다. </li></ol> 2. 림브 스킨 전체 - 마운트 Immunohistochemical 스테 이닝 <ol><li> MeOH / PBT (PBS + 0.2 % 트리톤 X - 100) (75 %, 50 %, 25 %) 5 분 각각의 등급 시리즈를 통해 잠복기에 의해 5ml 폴리 프로필렌 왕복 아래 튜브의 사지 스킨을 Rehydrate. 솔루션을 교환하면 사지 스킨 손상을 방지하기 위해주의를 기울여야합니다. </li><li> 상온에서 Nutator 믹서에서 부드러운 혼합과 PBT에서 5 분 두 번 씻으십시오. </li><li> Nutator 믹서에서 부드러운 혼합 2 시간 동안 당나귀 차 항체에 대한 10 %의 염소 차 항체에 대한 혈청 / PBS 0.2 % TX100 차단 버퍼 염소 또는 10% 돈키 혈청 / PBS 0.2 % TX100 차단 버퍼에서 하나와 사지 스킨 블럭 상온. </li><li> 차단 버퍼의 기본 항체의 800μl (테이블에 나열된 적절한 희석) (와 2ml - microcentrifuge 관에 고리 집게로 35 X 10mm 페트리 접시와 전송에 사지 스킨을 플레이스 중 10 % 염소 혈청 / PBS 0.2 % TX100 또는 10% 돈키 혈청 / PBS 0.2 % TX100). 4 ° C 하룻밤에 Nutator 믹서에 부드러운 믹싱으로 사지 스킨을 품어. 종 (예를 들어, 쥐의 단클론 항체 polyclonal 항체 + 토끼)에서 파생된 여러 기본 항체를 동시에 사용할 수 있습니다. </li><li> 다음날, 세척 버퍼의 4ml (중 2 %의 염소 혈청 / PBS 0.2 % TX100 또는 2 왕복 하단 튜브 5ml 폴리 프로필렌으로 고리 집게로 35 X 10mm 페트리 접시와 전송에 사지 스킨을 장소 % 동키 혈청 / PBS 0.2 % TX100). </li><li> 부드러운 실온에서 Nutator 믹서에서 혼합과 함께 15 분 다섯 번 씻으십시오. </li><li> 차단 버퍼 이차 항체의 800μl (중 10 % 염소 혈청 / PBS 0.2 % TX100하거나 10% 돈키 혈청 /를 함께 2ml - microcentrifuge 관에 고리 집게로 35 X 10mm 페트리 접시와 전송에 사지 스킨을 플레이스 PBS 0.2 % TX100). 일반적으로 우리는 잭슨 ImmunoResearch (Cy3, Cy5, 1:300 dilutions) 또는 Invitrogen (알렉사 플루어 488, 알렉사 플루어 568, 알렉사 플루어 633, 1:250 희석)에서 보조 항체를 사용합니다. 보조 항체의 집계 입자를 제거하는 0.22μm PVDF 막 주사기 필터를 사용하여 보조 항체 솔루션을 필터링합니다. 상온에서 Nutator 믹서에서 부드러운 혼합 1 시간 동안 어둠이나 알루미늄 호일로 싼에서 사지 스킨을 품어. 종에서 파생된 다른 형광 복합 이차 항체를 동시에 사용할 수 있습니다. </li><li> 세척 버퍼의 4ml (2 % 염소 혈청 / PBS 0.2 % 중 TX100 또는 2% 돈키 혈청 / PBS로 5ml 폴리 프로필렌 왕복 하단 튜브에 고리 집게로 35 X 10mm 페트리 접시와 전송에 사지 스킨을 플레이스 0.2 % TX100). 어둠이나 부드러운 실온에서 Nutator 믹서에서 혼합과 알루미늄 호일로 싸서 15 분 다섯 번 씻으십시오. </li><li> (핵에 대한 counterstaining에 대한 옵션)는 세척 버퍼의 4ml (중 2 %의 염소 혈청 / PBS 0.2 % TX100 또는 2% 돈키 혈청 / PBS 0.2 % TX100)에 - PRO - 3 (Invitrogen T3605과 함께 사지 스킨을 품어 , 어둠 또는 객실 temperatu에서 Nutator 믹서에서 부드러운 혼합 10 분 알루미늄 호일로 싼에서 1:3000 희석)다시. 그런 다음에 부드러운 혼합과 알루미늄 호일로 어둡거나 포장의 세척 버퍼의 4ml (2 % 염소 혈청 / PBS 0.2 % TX100 또는 2% 돈키 혈청 / PBS 0.2 % TX100을)를 5 분 세 번 씻어 상온에서 Nutator 믹서. 핵에 대한 강력하고 구체적인 얼룩이 특정 방출 (HeNe 633 나노미터 여기)에서하려면 - PRO - 3에 대한 관찰됩니다. </li></ol> 3. 슬라이드에 림브 스킨 장착 <ol><li> 35 X 10mm 페트리 접시에 사지 스킨을 놓습니다. 표백 광범위한 사진을 피하기 위해 낮은 조명과 stereomicroscope 이하 벌금 핀셋을 사용하여 스킨의 내부 계층에서 먼지, 크리스탈, 섬유를 제거합니다. </li><li> 고리 집게에 의해 접착제 현미경 슬라이드 사지 스킨을 전송합니다. 슬라이드 (coverslip으로 IE)에서 위쪽으로 누워 내부 레이어로 피부를 놓습니다. 신중하게 잘 핀셋을 사용하여 스킨을 평평하게하고 Kimwipe하여 버퍼를 세척 - 이월 제거하십시오. </li><li> 기포없이 안티 - 페이드 장착 매체 마운트합니다. 우리는 25 &quot;x25&quot;coverslip을 사용합니다. 어둠의 평평한 표면에 치료 (예 : 샘플보기 전에 실온에서 어둠 속에서 야간 배치 골드 시약을 연장 사용하여 마운트). 장기 저장을 위해, 4 슬라이드에 coverslip하고 저장을 날인 ° C. </li></ol> 4. 공촛점 현미경 <ol><li> fluorophores에 적합한 레이저를 설정합니다. 우리는 아르곤 488nm (알렉사 플루어 488와 GFP)을, DPSS 561nm (알렉사 플루어 568와 Cy3) 및 HeNe 633nm (알렉사 플루어 633, Cy5 및 - PRO - 3)을 포함하여 세 레이저 소스 Leica TCS SP5 공촛점 현미경을 사용하여 . </li><li> 더블 또는 트리플 묻은 샘플의 모든 염료가 동시에 흥분됩니다되는 누화를 방지하거나 줄이기 위해 순차적 스캔 도구를 사용하십시오. 순차 스캔 모드에서는, 이미지가 순차적으로 기록됩니다. </li><li> 여기에 대한 형광 염료와 레이저에 대한 더 일반적인 정보는 힙스 (2004)에 의해 &quot;생물학에 대한 공촛점 현미경&quot;의 설립 수 있습니다 </li></ol> 5. 대표 결과 팬 - 내피 세포 마커에 항체와 E15.5에서 마우스 forelimb의 피부에 전체 마운트 트리플 라벨 공촛점 immnofluorescence 현미경 PECAM - 1 (그림 1A - C, D 블루), neurofilament 마커 2H3 (그림 1A 녹색), 그리고 평활근 세포 마커 αSMA (그림 1A, B 적색)는 사지 피부에 2H3 + 주변 신경에 부합 αSMA + 동맥의 특성 분기 패턴을 공개했다. 분기 혈관뿐만 아니라,이 사지 피부 vasculature 모델은 림프 내피 세포 마커 LYVE - 1 (그림 1D, E 적색)와 Neuropilin2 (NRP2​​) (그림 1D, F 녹색으로 항체를 사용하여 분기 림프 혈관의 patterning을 연구하는 데 사용됩니다 ). <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="그림 1" /> 그림 1. (AC) 동맥은 태아의 사지 피부에 말초 신경로 맞춥니다. 팬 - 내피 세포 마커 PECAM - 1 (AC, 파랑), neurofilament 마커 2H3 (A, 녹색), 그리고 평활근 세포 마커 αSMA (A와 B, 빨간색으로 항체와 함께 전체 마운트 트리플 라벨 공촛점 immnofluorescence 현미경 )이 표시됩니다. E15.5, 2H3 +에서 말초 신경 (오픈 화살표) αSMA에 의해 덮여 동맥 (화살촉) + 평활근 세포와 연결합니다. 배아 사지 피부에 (DF) 림프 vasculature. 팬 - 내피 세포 마커에 항체 PECAM - 1 (D, 파랑), 림프 내피 세포 마커 LYVE - 1 (D와 E, 빨간색)와 Neuropilin2 (NRP2​​) (D와 F, 녹색과 함께 트리플 라벨 공촛점 immnofluorescence 현미경 )이 표시됩니다. LYVE - 1도 macrophages (오픈 화살촉)의 하위 집합에 의해 표현된다 반면에 림프 혈관은 LYVE - 1 NRP2 모두에 의해 시각입니다. 스케일 바 : 100μm.

<ol>
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관심 없음 충돌 선언하지 않습니다.

그것이 충분한 산소와 장기에 영양분을 공급하기 때문에 혈관 시스템은 embryogenesis 동안뿐만 아니라 성인의 장기 유지 보수 및 생식 기능에 대한 장기 개발을 위해 매우 중요합니다. 적절한 혈관 네트워크는 기존의 모세관이 높은 분기와 계층 구조를 개편하고있는 angiogenesis에 의해 복잡하고 여러 단계의 프로세스를 잘 설정됩니다. 다수의 작품이 분자의 다양한 이러한 과정에 관여하는 것으로 나...

항체 판 - 내피 세포 마커 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> PECAM - 1 </td><td> 아르메니아어 햄스터 (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> 1:100 희석 # 1 </td></tr><tr><td> PECAM - 1 </td><td> 랫 (M) </td><td> BD </td><td> 553369 </td><td> 1:300 희석 </td></tr><tr><td> VEGFR2 </td><td> 랫 (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> 1:200 희석 </td></tr><tr><td> CD34 </td><td> 랫 (M) </td><td> eBioscience </td><td> 13-0341 </td><td> 1:300 희석 </td></tr><tr><td> 콜라겐 IV </td><td> 래빗 (P) </td><td> AbD Serotec </td><td> 2150년부터 1470년까지 </td><td> 1:300 희석 # 2 </td></tr></tbody></table> 아티 내피 세포 마커 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> Neuropilin1 </td><td> 래빗 (P) </td><td> 알렉스 Kolodkin의 실험실 # 3 </td><td></td><td> 1:3000 희석 </td></tr><tr><td> Unc5H2 </td><td> 염소 (P) </td><td> R &amp; D </td><td> AF1006 </td><td> 1:200 희석 </td></tr></tbody></table> 정맥 내피 세포 마커 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> EphB4 </td><td> 염소 (P) </td><td> R &amp; D </td><td> AF446 </td><td> 1:100 희석 </td></tr></tbody></table> 림프 내피 세포 마커 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> LYVE - 1 # 4 </td><td> 래빗 (P) </td><td> Abcam </td><td> ab14917 </td><td> 1:200 희석 </td></tr><tr><td> LYVE - 1 # 4 </td><td> 랫 (M) </td><td> MBL </td><td> D225 - 3 </td><td> 1:200 희석 </td></tr><tr><td> Prox - 1 </td><td> 래빗 (P) </td><td> Chemicon </td><td> AB5475 </td><td> 1:1000 희석 </td></tr><tr><td> Prox - 1 </td><td> 염소 (P) </td><td> R &amp; D </td><td> AF2727 </td><td> 1시 50분 희석 </td></tr><tr><td> Neuropilin2 </td><td> 래빗 (P) </td><td> 세포 신호 </td><td> 3366 </td><td> 1, 100 희석 </td></tr><tr><td> Podoplanin </td><td> 시리아 햄스터 (M) </td><td> 하이 브리 도마 뱅크 </td><td> 8.1.1 </td><td> 1:200 희석 </td></tr></tbody></table> 평활근 세포 / pericyte 마커 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> αSMA - CY3 </td><td> 마우스 (M) # 5 </td><td> 시그마 </td><td> C - 6198 </td><td> 1:500 희석 # 6 </td></tr><tr><td> NG2 </td><td> 래빗 (P) </td><td> Chemicon </td><td> AB5320 </td><td> 1:200 희석 </td></tr><tr><td> SM22α </td><td> 래빗 (P) </td><td> Abcam </td><td> ab14106 </td><td> 1:200 희석 </td></tr></tbody></table> GFP 기자에 대한 항체 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> GFP </td><td> 래빗 (P) </td><td> Invitrogen </td><td> A11122 </td><td> 1:300 희석 </td></tr><tr><td> GFP </td><td> 랫 (M) </td><td> Nacalai tesque </td><td> 04404-84 </td><td> 1:1000 희석 </td></tr><tr><td> GFP </td><td> 칙 (P) </td><td> Chemicon </td><td> P42212 </td><td> 1:300 희석 </td></tr></tbody></table> LacZ 기자에 대한 항체 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> β - 여자 </td><td> 래빗 (P) </td><td> MP 바이오 메디컬 </td><td> 55,976 </td><td> 1:5000 희석 </td></tr><tr><td> β - 여자 </td><td> 염소 (P) </td><td> AbD Serotec </td><td> 4600-1409 </td><td> 1:500 희석 </td></tr><tr><td> β - 여자 </td><td> 칙 (P) </td><td> Abcam </td><td> ab9361 </td><td> 1:200 희석 </td></tr></tbody></table> 주변 축삭에 대한 항체 <table border="1"><tbody><tr><td> 항독소 </td><td> 종 </td><td> 회사 </td><td> 카탈로그 # </td><td> 근무 조건 </td></tr><tr><td> 2H3 </td><td> 마우스 (M) # 7 </td><td> 하이 브리 도마 뱅크 </td><td> 2H3 </td><td> 1:200 희석 </td></tr><tr><td> Tuj1 </td><td> 마우스 (M) # 8 </td><td> Covance </td><td> MMS - 435P </td><td> 1:500 희석 </td></tr><tr><td> Peripherin </td><td> 래빗 (P) </td><td> Chemicon </td><td> AB1530 </td><td> 1:1000 희석 </td></tr></tbody></table> 마이 그 레이션 Schwann 세포에 대한 항체 <table border="1"><tbody><tr><td> BFABP </td><td> 래빗 (P) </td><td> 토마스 뮐러의 실험실 # 9 </td><td></td><td> 1:3000 희석 </td></tr></tbody></table> (P) : polyclonal 항체 (M) : 단클론 항체 # 1 : 염소 방지 아르메니아어 햄스터 - Cy3 (잭슨 ImmunoResearch 127-165-160) 항체가 보조 항체로 사용해야합니다. # 2 : 콜라겐 IV 항체가 원위치 하이브리드화 이후 혈관을 감지하는 데 사용할 수 있습니다. # 3 : Neuropilin1 항체가 친절하게 존스 홉킨스 대학의 알렉스 Kolodkin의 실험실에 의해 제공됩니다. 우리는 아직 테스트하지 않았습니다 있지만, 양 안티 인간 Neuropilin1 항체는 R &amp; D (AF3870)에서 사용할 수 있습니다. # 4 : LYVE - 1 항체는 또한 배아 피부에 macrophages의 하위 집합을 감지합니다. # 5 : 안티 αSMA 항체 마우스 IgG2a 단클론 항체이다. # 6 : Cy3 - 복합 αSMA 항체는 초와 함께 실온에서 1 시간 incubated입니다다른 일차 항체에 대한 항체 ondary. # 7 : 2H3 항체가 neurofilament에 대한 마우스 IgG1 단클론 항체이다. # 8 : Tuj1 항체가 신경 세포 특정 클래스 III 베타 tubulin에 대한 마우스 IgG2a 단클론 항체이다. # 9 : BFABP (두뇌 특정 지방산 결합 단백질) 항체가 친절 분자 의학에 대한 최대 - Delbrück - 센터의 토마스 뮐러의 실험실에 의해 제공됩니다.

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

이미지에 대한 전체 마운트 immunohistochemical 분석은 전체 vasculature는 morphogenesis을 분기의 세포 메커니즘을 이해하는 데 필수적입니다. 우리는 기존의 원시 모세관 신경 얼기가 계층 브랜츠드 혈관 네트워크에 개편되는 혈관 개발을 연구하기 위해 사지 피부 vasculature 모델을 개발했습니다. 여러 개의 라벨과 함께 전체 - 마운트 공촛점 현미경 특정 형광 마커를 사용하여 내피 세포, pericytes 및 평활근 세포를 포함하여 그대로 혈관뿐만 아니라 자신의 세포 구성 요소의 강력한 이미징을 허용합니다. 유전자 연구와 함께 사지 피부 vasculature 모델의 발전은 혈관 개발 및 patterning의 분자 메커니즘을 이해하고 개선합니다. 사지 피부 vasculature 모델은 말초 신경이 동맥의 차별과 patterning을위한 공간 템플릿을 제공하는 방법을 연구하는 데 사용되었습니다. 이 동영상이 문서에서는 우리가 혈관 개발의 과정에 따라 수 있습니다 vascularized 배아 기관에 대한 적용 혈관 특정 항체 및 형광 항체 보조와 완전한 혈관을 얼룩이 간단하고 강력한 프로토콜을 설명합니다.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

우리는 전체 마운트 immunohistochemistry를 소개하고 레이저 마우스 배아 사지 피부에 복잡한 혈관 네트워크 형성을 분석하는 데 여러 상표와 공촛점 현미경을 검사합니다.

배아 림브 스킨 Vasculature에 대한 전체 마운트 Immunohistochemical 분석 : 엠브료에 혈관 분기 Morphogenesis를 공부하는 모델 시스템

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K 조회수.  National Heart, Lung, and Blood Institute, National Institutes of Health. 우리는 전체 마운트 immunohistochemistry를 소개하고 레이저 마우스 배아 사지 피부에 복잡한 혈관 네트워크 형성을 분석하는 데 여러 상표와 공촛점 현미경을 검사합니다.

비디오: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

연구

생물학

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

칙의 전체 마운트 항체 스테 이닝을위한 방법

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish 전체 마운트 고해상도 더블 형광등 현장에서 하이브 리다이 제이션

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

마우스 배아의 폐 문화, 분기의 분자 메커니즘을 평가하는 시스템

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

무 혈청 전체 배아 배양 시스템에서 마우스 배아 개발

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

사용<em&gt; 예쁜 꼬마 선충</em&gt; 다세포 생물의 단백질 항상성을 연구하는 모델 시스템으로

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...