저자는 Leif Oxburgh와 Derek Adams에게 도움을 주신 Leif Oxburgh와 Stefan Wölfl 및 Ulrike Müller와 Saskia Schmitteckert, Julia Gobbert, Sascha Weyer 및 Viola Mayer에게 도움을 주신 데 대해 감사드립니다. 랩. 이 작품은 개발, 생물 학자의 회사 (CP) 에 의해 지원되었다 .

마우스는 스웨덴의 규정 및 EU 법령 (2010 / 63 / EU)에 따라 유지 관리되었습니다. 모든 절차는 스웨덴 윤리위원회 (C79 / 9, C248 / 11 및 C135 / 14 허가)의 지침에 따라 수행되었습니다. 동물과 관련된 하이델베르그 대학의 절차는 하이델베르그 대학의 Regierungspräsidium Karlsruhe와 Animal Welfare Officers에 의해 승인되었습니다. 1. 시약 및 시약의 준비 참고 : 오염을 최소화하려면 층류 후드를 사용하십시오. <ol><li> 해부 당일에, 무균 인산 완충 식염수 (PBS) 중의 1 : 5의 몰비로 항 - 인간 Fc 염소 항체와 혼합함으로써 인간 Fc 단독 또는 인간 Fc에 융합 된 재조합 키메라 에프 린 단백질을 사전 - 클러스터링한다. 37 ° C에서 1 시간 동안 품어 둡니다. 5 . </li><li> Dulbecco &#39;s Modified Eagle Medium : Nutrient를 보충하여 배양 배지를 준비하십시오.1 % 페니실린 / 스트렙토 마이신 (v / v), 1 % 글루타민 (v / v), 1 % 인슐린 - 트랜스페린 - 셀레늄 (v / v), 50 μg / ml 인간 홀로 코 - 트랜스페린 및 1.5㎍ / mL 암포 테리 신이 포함된다. 참고 : 16 ML 배아의 10 ML 충분합니다. </li><li> 배양액에 최종 농도 20 μg / mL의 클러스터 된 재조합 에프린 용액을 첨가합니다. 클러스터 된 인간 Fc를 대조군으로 사용하십시오. </li><li> 무균, 코팅되지 않은 평평한 바닥 폴리스티렌 4 잘 접시의 각 우물에 문화 매체 500 μL를 추가하여 문화 플레이트를 준비합니다. </li><li> 무균 포셉을 사용하여 신중하게 매체 상단에 구멍 당 5 μm의 기공 크기와 하나의 폴리 카보 네이트 멤브레인 필터를 놓으십시오. 인큐베이터 (37 ° C / 5 % CO 2 )에 준비 접시를 전송하십시오. 필터가 윗면이 마를 때까지 뜨고 플로트가 발생하는지 확인하십시오. 참고 : 두 개의 신장 혈관에 대해 하나의 필터로 충분합니다. </li><li> 면도날을 사용하여 대략 30 피펫 팁 (볼륨 : 100 μL) approximately 끝에서 1mm가 더 넓게 열립니다. 팁을 다시 피펫 - 팁 랙에 넣으십시오. </li></ol> 2. E11.5에서 Metanephric Rudiments의 해부 <ol><li> 자궁 경부 탈구 (또는 지역 윤리위원회의 승인을받은 방법을 사용)하여 E11.5에서 임신 임신 한 마우스를 희생합니다. </li><li> Brown 외 6 에 설명 된대로 자궁과 배아를 제거합니다. 이 시점에서 해부 현미경으로 작업하십시오. </li><li> 5 번 시계 제조기 겸자를 사용하여 앞다리 바로 아래에서 배아를 잘라냅니다. 각 배아에 신선한 페트리 접시를 사용하십시오. 배아의 꼬리와 다리를 제거하십시오. 이렇게하려면 5 번 시계 제조기 집게 한 쌍으로 제거 할 조직을 잡고 두 번째 쌍의 끝을 사용하여 자릅니다. </li><li> 복부 기관의 질량을 제거하려면, 꼬리의 주둥이 방향으로 배아의 몸 벽을 옆으로 열려면 한 쌍의 포셉의 팁을 사용하십시오. <ol><li> 외형 적으로 절단, t와 평행하게그는 지느러미 대동맥. 오리 엔테이션을 위해 혈액으로 채워지기 때문에 눈에 보이는 대동맥을 사용하십시오. 조심스럽게 폐쇄 포셉의 팁을 사용하여 등쪽 부분에서 신체 벽의 복부 부분을 분리하고 배아를 뒤집습니다. 앞면과 마찬가지로 몸통 벽을 옆으로 대동맥 - 꼬리 방향으로 절단하고 배부 대동맥을 사용하여 방향을 잡습니다. </li></ol></li><li> 복부 몸의 벽이 지느러미 몸체에서 분리되면, 한 쌍의 포셉으로 잡아서 신중하게 잡아 당겨 기관 덩어리와 함께 제거하십시오. 참고 : metanephric anlagen은 일반적으로 등쪽 몸 벽에 붙어있어. 그들은 타원형이며, 뒷다리 수준에 위치한 ~ 200 μm 길이의 구조물입니다. </li><li> 복부 쪽이 위로 향하게 한 쌍의 포 셉를 사용하여 등쪽 몸통 벽을 지킨다. 포셉의 다른 한 켤레를 사용하여, 그것의 주둥이 끝에서 등쪽 대동맥을 잡고 조심스럽게 몸의 벽에서 등 대동맥을 껍질. 참고 : 두 metanephric 신장은 일반적으로 붙어있어등 대동맥에 붙어있다. </li><li> 한 쌍의 포셉을 사용하여 신장 anlagen에 rostrally 잘라 대동맥을 제거하십시오. 그런 다음 신중하게 포 셉의 팁을 사용하여 두 신장 anlagen 분리하십시오. </li><li> 조심스럽게 포셉의 팁을 사용하여 신장 anlagen에서 원치 않는 조직을 껍질. mesenchyme을 손상시키지 않도록주의하십시오. 손상은 추가 분석에서 explant의 성장 및 배제를 초래할 수 있습니다. </li><li> 4 잘 접시에 폴리 카보 네이트 멤브레인 필터에 PBS 10 μL에 microliter 피펫과 와이드 오픈 피펫 팁을 사용하여 신장 anlagen을 별도로 전송합니다. 참고 : 표면 장력으로 인해 체외 이식편은 얇은 매질로 덮여 있습니다. 하나의 필터로 두 개의 신장 혈관이 충분합니다. <ol><li> 두 개의 신장 anlagen 각 필터의 ​​각 절반의 중심에 놓습니다. 37 ° C / 5 % CO 2 에서 배양기로 플레이트를 다시 옮긴다. metanephric rud 때까지 인큐베이터에 접시를 남겨주세요다음 배아에서 iments은 필터에 배치 할 준비가되었습니다. </li></ol></li><li> 각 배아에 대해 2.3-2.9.1 단계를 반복합니다. 참고 : 일부 연습으로 해부 절차는 배아당 약 5 분 걸립니다. </li><li> 37 ° C / 5 % CO 2 에서 3 일 동안 신장 anlagen을 품어. </li></ol> 3. 고정 및 염색을위한 시약의 준비 <ol><li> 칼슘과 마그네슘 (w / v)이없는 PBS에서 4 % 파라 포름 알데히드 (PFA)를 준비하려면 PFA를 60 ℃에서 PBS에 녹여서 지속적으로 저어줍니다. 실온으로 식힌 다음 페이퍼 필터를 사용하여 남아있는 입자를 제거합니다. 분주하고 4 ° C에서 보관하여 즉시 사용하거나 장기간 보관할 수 있습니다. 주의 : PFA는 유독합니다. 지역 안전 표준에 명시된 개인 보호 장비를 착용하고 흄 후드에서 작업하십시오. 시설 지침에 따라 지정된 폐기물 용기를 사용하여 폐기물을 버립니다. </li><li> permeabilization solution을 준비하기 위해 희석 된 Triton X-1000.3 % (v / v)가되도록 칼슘과 마그네슘이없는 PBS에서 처리 하였다. </li><li> 차단 용액을 준비하기 위해 5 % (v / v) 염소 혈청과 0.1 % Triton X-100을 칼슘과 마그네슘없이 PBS에 첨가한다. 나트륨 아자 이드를 0.02 % (w / v)의 최종 농도로 첨가한다. 4 ° C에서 보관하십시오. 주의 : 나트륨 아자 이드는 독성이 있습니다. 지역 안전 및 환경 보호 규정에 따라 나트륨 아자 이드를 취급하십시오. 참고 : 염색을 위해 항체를 사용하는 경우 2 차 항체가 유도 된 종에서 5 % 혈청을 사용하여 차단 용액을 만듭니다. </li><li> biotinylated Dolichorus biflorus agglutinin을 1 : 200으로 블로킹 용액으로 희석한다. Alexa488 - conjugated streptavidin을 1 : 200으로 블로킹 용액으로 희석하십시오. </li><li> 즉시 사용할 수있는 배지 (물질 표 참조)를 만들기 위해 0.1M Tris-HCl (pH 8.5)에 녹인 25 % (w / v) 글리세롤에 0.1g의 수용성 폴리 비닐 알코올 계 점착제 / mL를 넣는다. . 연속 교반하에 50 ° C로 1 시간 가열하고 식힌 다음 1을 사용하여 pH를 8.0-8.5로 조정한다.M NaOH. NaOH 농도를 높이 지 마십시오. 0.02 % (w / v)의 최종 농도로 100 μg / mL의 N- 프로필 게 네이트와 티 메로 살을 첨가한다. 실온에서 30 분 동안 교반한다. 주의 : Thimerosal은 독성이 있습니다. 해당 지역의 안전 및 환경 보호 규정에 따라 취급하십시오. <ol><li> 50 ML 튜브에 삽입 매체를 채우고, 로터를 균형을, 그리고 3,200 XG 및 10 분 실온에서 원심 분리기. 15 mL 튜브에 투명한 용액을 따라 내고 펠렛을 버린다. 분주하고 -20 ℃에서 몇 주 동안 보관하십시오. 해동되면 용액을 4 ° C에 유지한다. 사용 직전 ~ 30 ° C로 예열하십시오. </li></ol></li><li> explants와 필터가 장착 될만큼 많은 유리 슬라이드를 준비합니다. 이를 위해 인스턴트 접착제를 사용하여 유리 슬라이드 양쪽에 18 x 18 mm 크기의 작은 커버 슬립 2 개를 붙이고 필터 사이에 15 mm의 공간을 남겨 둡니다. </li></ol> 4. 고정 및 염색 <ol><li> 배양 기간 3배양기에서 플레이트를 제거하고 조심스럽게 micropipette를 사용하여 각 우물에서 문화 매체를 대기음. 필터를 만지지 말고 팁 입구를 우물 벽쪽으로 유지하십시오. </li><li> 각 우물에 4 % PFA / PBS 500 μL를 추가합니다. 필터는 정착액 위에 뜨게됩니다. 마이크로 피펫을 사용하여 PFA 고정액을 조심스럽게 첨가하여 필터를 잠수시킵니다. 실온에서 30 분 동안 품어 낸다. 참고 : 다음 단계를 수행 할 때 필터에서 외과 체를 씻어 내지 않도록주의해야합니다. 우물 벽을 향한 피펫 팁의 개방을 유지하십시오. </li><li> PFA를 제거하고 500 μL의 PBS로 두 번 헹구십시오. 각 우물에 permeabilization 솔루션 500 μL를 추가하고 1 시간 동안 실온에서 품어. </li><li> PBS 500 μL로 두 번 씻어서 각 잘 차단 솔루션의 500 μL를 추가합니다. 실온에서 1 시간 동안 인큐베이션한다. </li><li> 차단 용액을250 μL의 biotinylated Dolichorus biflorus agglutinin은 1 : 200으로 블로킹 용액으로 희석하고 4 ° C에서 24 시간 동안 배양한다. </li><li> Dolichorus biflorus agglutinin 솔루션을 제거하고 잘 PBS 당 500μL로 씻어. </li><li> Alexa488 - conjugated streptavidin 250 μL를 1 : 200 희석 용액에 첨가하고 4 ° C에서 24 시간 동안 배양하십시오. 또는 실온에서 2 시간 동안 배양하십시오. 참고 : 4 ° C에서 배양하면 더 나은 신호 대 잡음비가 달성됩니다. </li><li> 1 웰당 PBS 500 μL로 두 번 씻어. 집게를 사용하여 explants이 위로 향하게 준비 슬라이드에 필터를 전송합니다. 임베딩 매체 ~ 50-100 μL로 탑재하십시오. 60mm 길이의 1.5 번 커버 슬립으로 덮으십시오. 매질 용액을 실온에서 2 시간 동안 응고시킨다. 이미지 준비가 끝날 때까지 4 ° C에서 포일로 싸인 슬라이드를 이미지화하거나 보관하십시오. </li><li> 와이드 필드 epifluorescence 현미경 7 &lt;/ sup&gt;를 Hg- 램프에 연결하고 청색 여기 (여기 대역 통과, 460 - 495 nm, 이색 거울, 505 nm, 방출 대역, 510 - 550 nm) 및 20X Plan-Apochromat 렌즈 용 거울 장치를 사용하여 0.75 구멍. </li></ol>

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(50), e2555 (2011).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+interaction+in+the+development+of+the+mouse+metanephros.">Inductive interaction in the development of the mouse metanephros.</a> J Exp Zool. 130, 319-340 (1955).</li><li>Sax&#233;n, L., Toivonen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primary Embryonic Induction. , (1962).</li><li>Sax&#233;n, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J., Wartiovaara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+kidney+mesenchyme+in+an+experimental+model+system.">Differentiation of kidney mesenchyme in an experimental model system.</a> Adv Morphog. 7, 251-293 (1968).</li><li>Dudley, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+FGF+and+BMP+signaling+pathways+regulates+development+of+metanephric+mesenchyme.">Interaction between FGF and BMP signaling pathways regulates development of metanephric mesenchyme.</a> Genes Dev. 13, 1601-1613 (1999).</li><li>Per&#228;l&#228;, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sema4C-Plexin+B2+signalling+modulates+ureteric+branching+in+developing+kidney.">Sema4C-Plexin B2 signalling modulates ureteric branching in developing kidney.</a> Differentiation. 81 (2), 81-91 (2011).</li><li>Thesleff, I., Ekblom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+transferrin+in+branching+morphogenesis,+growth+and+differentiation+of+the+embryonic+kidney.">Role of transferrin in branching morphogenesis, growth and differentiation of the embryonic kidney.</a> J Embryol exp Morph. 82, 147-161 (1984).</li><li>Watanabe, T., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+ureteric+bud+branching+morphogenesis.">Real-time analysis of ureteric bud branching morphogenesis.</a> Dev Biol. 271, 98-108 (2004).</li><li>Sebinger, D. D. R., Unbekandt, M., Ganeva, V. V., Ofenbauer, A., Werner, C., Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel,+Low-Volume+Method+for+Organ+Culture+of+Embryonic+Kidneys+That+Allows+Development+of+Cortico-Medullary+Anatomical+Organization.">A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization.</a> PLoS One. 5 (5), e10550 (2010).</li><li>Ekblom, P., Miettinen, A., Virtanen, I., Wahlstr&#246;m, T., Dawnay, A., Sax&#233;n, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+segregation+of+the+metanephric+nephron.">In vitro segregation of the metanephric nephron.</a> Dev Biol. 84 (1), 88-95 (1981).</li><li>Davies, J. A., Unbekandt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA-mediated+RNA+interference+in+embryonic+kidney+organ+culture.">siRNA-mediated RNA interference in embryonic kidney organ culture.</a> Methods Mol Biol. 886, 295-303 (2012).</li><li>Sax&#233;n, L., Lehtonen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+kidney+in+organ+culture.">Embryonic kidney in organ culture.</a> Differentiation. 36 (1), 2-11 (1987).</li><li>Bard, J. B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+mouse+kidney+embryogenesis+writ+small.">The development of the mouse kidney embryogenesis writ small.</a> Curr Opin Genet Dev. 2, 589-595 (1992).</li><li>Davies, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments.">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li><li>Batourina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret.">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li></ol>

저자는 공개 할 것이 없습니다.

이 원고는 마우스 배아에서 발생하는 metanephric anlagen을 분리하고 장기 기초를 배양하는 방법을 설명합니다. 이 방법은 Grobstein 8 과 Saxén 9 , 10 에 의해 개발 된 표준 기법이며 많은 다른 사람들에 의해 수정되고 수정되었습니다 11 , 12 . 이 방법의 성공은 주로 절개의 지속 기간과 장기간의 절?...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, calcium, magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14040117</td>
			<td>use for dissection</td>
		</tr>
		<tr>
			<td>holo-Transferrin human</td>
			<td>Sigma-Aldrich</td>
			<td>T0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS -G) (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B solution</td>
			<td>Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>Sigma-Aldrich</td>
			<td>T5125</td>
			<td></td>
		</tr>
		<tr>
			<td>Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>2370</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma-Aldrich</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Dolichorus Biflorus Agglutinin</td>
			<td>Vector Laboratories</td>
			<td>B-1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa488 conjugated Streptavidin</td>
			<td>Jackson Immuno Research</td>
			<td>016-540-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>496-EB</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IgG1 Fc, CF</td>
			<td>R&#38;D Systems</td>
			<td>110-HG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Human IgG Fc Antibody</td>
			<td>R&#38;D Systems</td>
			<td>G-102-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline tablets</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td>use for fixation and immunostaining</td>
		</tr>
		<tr>
			<td>Dumont #5, biologie 
			tips, INOX, 11cm</td>
			<td>agnthos.se</td>
			<td>0208-5-PS</td>
			<td>2 pairs of forceps are needed</td>
		</tr>
		<tr>
			<td>Iris scissors, straight, 12cm</td>
			<td>agnthos.se</td>
			<td>03-320-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps, 
			straight, delicate, 13cm</td>
			<td>agnthos.se</td>
			<td>08-032-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes Nunclo Delta treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>150679</td>
			<td></td>
		</tr>
		<tr>
			<td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &#181;m, 13 mm, white, plain</td>
			<td>MerckMillipore</td>
			<td>TMTP01300</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon Multidishes 
			4 wells, flat bottom</td>
			<td>Sigma-Aldrich</td>
			<td>D6789-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass24x50mm thickn. No.1.5H 0.17+/-0.005mm</td>
			<td>nordicbiolabs</td>
			<td>107222</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses No.1.5, 18x18mm</td>
			<td>nordicbiolabs</td>
			<td>102032</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides ~76x26x1, 1/2-w. ground plain</td>
			<td>nordicbiolabs</td>
			<td>1030418</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Razor Blades</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430055</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1213125</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed stage research mircoscope</td>
			<td>Olympus</td>
			<td>BX61WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice, male, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 6 inbred mice,female, C57BL/6NTac</td>
			<td>Taconic</td>
			<td>B6-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Greenough Stereo Microscope</td>
			<td>Leica</td>
			<td>Leica S6 E</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and culture of mouse embryonic kidney

이 프로토콜의 목표는 마우스 metanephric 기초의 해부, 격리 및 문화에 대한 방법을 설명하는 것입니다. 포유류 신장이 발달하는 동안 두 개의 전구 세포 조직, 즉 ureteric bud와 metanephric mesenchyme은 세포 기작을 전달하고 상호 작용하여 결국 포집 시스템과 신장의 네프론을 형성합니다. 포유류 배아가 자궁 내에서 자라며 관찰자가 접근하기 어렵 기 때문에 장기 배양이 개발되었습니다. 이 방법을 사용하면 신장 조직 형성 과정에서 상피 간엽 상호 작용과 세포 행동을 연구 할 수 있습니다. 또한 선천성 신장 및 비뇨 생식기 기형의 기원을 조사 할 수 있습니다. 조심스럽게 해부 된 후, metanephric 기초 물질은 배양 배지에 떠있는 필터로 옮겨져 며칠 동안 세포 배양기에 보관 될 수 있습니다. 그러나 조건은 다음과 같습니다.인공이며 조직의 신진 대사에 영향을 줄 수 있습니다. 또한, 시험 물질의 침투는 체외 이식편에 존재하는 세포 외 기질 및 기저막으로 인해 제한 될 수있다. 기관 문화의 한 주요 이점은 실험자가 기관에 직접 접근 할 수 있다는 것입니다. 이 기술은 저렴하고 간단하며 생물학적 활성 물질의 첨가, 유전 변이의 연구, 진보 된 영상 기술의 적용과 같은 많은 수정을 가능하게합니다.

Metanephric kidn anlagen은 E11.5에서 임신 한 Black-6 근친 교배 마우스에서 추출하여 배양 하였다. 3 일 후, ureteric bud는 5 번까지 분지되었고, 그 결과 초기 T 자 형태의 ureteric bud가 분출되었다. 각 체외 이식편을 촬영하고 분지 생성량을 결정하고 분지 당 종말점 수를 계산하기 위해 분절 수와 종점을 정량화했습니다 ( 그림 1 ). ImageJ ( 제 4 세대), 제 4 세대는 처...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

이 프로토콜은 마우스 배아에서 metanephric의 기초를 분리하고 배양하는 방법을 설명합니다.

마우스 배아 신장의 해부 및 배양

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

11.8K 조회수.  Uppsala University.  이 프로토콜은 마우스 배아에서 metanephric의 기초를 분리하고 배양하는 방법을 설명합니다. 

비디오: Dissection and Culture of Mouse Embryonic Kidney

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

전사 분석을위한 대규모 Zebrafish의 배아 심장의 해부

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

연구

발생학

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Electroporation에 의한 배아 마우스 달팽이의 Explants의 문화 유전자 전송

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

문화 공동 문화 마우스의 난소와 난소

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

혈청이없는 단층 문화 마우스 배아 줄기 세포의 신경 분화

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

의 사체 프로파일 링<em&gt; 생체</em2 색의 마이크로 어레이 플랫폼을 사용하여&gt; 제작 소 착상 전 배아

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

쥐 배아 중족골의 문화 : 연골 골화의 생리 학적 모델

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

해 부 및 마우스 뇌 개발의 코로나 슬라이스 준비

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

해 부와 초파리 번데기 난소의 얼룩

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Organotypic 문화 메서드 배아 치킨 조직 개발 연구를

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

신경 문장 세포 박리 및 이동의 연구를 위한 마우스에서 1 차적인 두개골 신경 문장 세포의 해부, 문화 및 분석

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...