Name: determining bile duct density in the mouse liver
Uploaded: 2019-04-30T14:45:00
Description: Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

저자는 국립 보건원 (NIH)의 지원을 인정 (R01 GM084135 및 R01 DK109982), NIH P30 DK56338에서 텍사스 의료 센터 소화기 질환 센터에서 파일럿 / 타당성 상, 그리고 알라질 증후군 가속기 상에서 의료 재단.

모든 동물은 기관 동물 관리 및 사용 위원회 지침및 승인된 동물 프로토콜에 따라 베일러 의과 대학의 장벽 동물 시설에 보관되었습니다.
1. 마우스 간 조직의 컬렉션
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간 수확을위한 마우스의 준비
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<li>이소플루란을 사용하여 마우스를 안락사시.</li>
		<li>죽음을 보장하기 위해 마우스의 자궁 경부 탈구를 수행합니다.</li>
		<li>흉곽 아래 약 1인치 의 가?...

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M., 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=Features+of+Alagille+syndrome+in+92+patients:+frequency+and+relation+to+prognosis.">Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.</a> Hepatology. 29 (3), 822-829 (1999).</li><li>Poncy, A., 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=Transcription+factors+SOX4+and+SOX9+cooperatively+control+development+of+bile+ducts.">Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.</a> Dev Biol. 404 (2), 136-148 (2015).</li><li>Hofmann, J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+intrahepatic+biliary+tree.">Development of the intrahepatic biliary tree.</a> Semin Liver Dis. 22 (3), 213-226 (2002).</li><li>Kaneko, K., Kamimoto, K., Miyajima, A., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+remodeling+of+the+biliary+architecture+underlies+liver+homeostasis.">Adaptive remodeling of the biliary architecture underlies liver homeostasis.</a> Hepatology. 61 (6), 2056-2066 (2015).</li><li>Schaub, J. R., 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=De+novo+formation+of+the+biliary+system+by+TGFbeta-mediated+hepatocyte+transdifferentiation.">De novo formation of the biliary system by TGFbeta-mediated hepatocyte transdifferentiation.</a> Nature. 557 (7704), 247-251 (2018).</li><li>Sparks, E. E., Huppert, K. A., Brown, M. A., Washington, M. K., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+regulates+formation+of+the+three-dimensional+architecture+of+intrahepatic+bile+ducts+in+mice.">Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.</a> Hepatology. 51 (4), 1391-1400 (2010).</li><li>Tanimizu, 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=Intrahepatic+bile+ducts+are+developed+through+formation+of+homogeneous+continuous+luminal+network+and+its+dynamic+rearrangement+in+mice.">Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.</a> Hepatology. 64 (1), 175-188 (2016).</li><li>Walter, T. J., Sparks, E. E., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-dimensional+resin+casting+and+imaging+of+mouse+portal+vein+or+intrahepatic+bile+duct+system.">3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system.</a> J Vis Exp. (68), e4272 (2012).</li><li>Popper, H., Kent, G., Stein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductular+cell+reaction+in+the+liver+in+hepatic+injury.">Ductular cell reaction in the liver in hepatic injury.</a> J Mt Sinai Hosp N Y. 24 (5), 551-556 (1957).</li><li>Yimlamai, D., 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=Hippo+pathway+activity+influences+liver+cell+fate.">Hippo pathway activity influences liver cell fate.</a> Cell. 157 (6), 1324-1338 (2014).</li></ol>

마우스에서 BD 발달 및 수리의 분석은 담대성 무질서의 병인 그리고 기계장치를 공부에 있는 중요한 공구입니다. 또한, 새로운 치료법의 개발은 부분적으로 재현가능하고 바람직하게는 정량화 가능한 표현형을 확립하는 데 의존한다. 마우스 모형에 있는 현재 표현형은 일반적으로 세포 모형 특정 마커를 위한 혈청 화학, 간 신체 학 및 면역 염색을 관련시킵니다. 이러한 기술은 담즙 시스템의 구?...

담즙 나무는 포유류 간에서 중요 한 부분, 창 자에 간 세포에서 담 즙의 통과 허용. 간간 간 담관 (BDs)은 융기양양에 의해 형성되며, 이는 노치 및 TGFβ 신호전달을통해 이중 전위 간폭발과 구별되는 1,2. 성숙한 BD에 담관과 그들의 어셈블리의 적당한 명세서 그리고 투입은 간 간 담즙 나무의 발달을 위해 중요합니다. 간 개발 또는 장기 재생 시 성장, 담 즙 시스템 적절 한 담 즙 배수를 보장 하기 위해 간 따라 개발 해야. 더욱이, 많은 신디로믹 및 비 신드로믹 질환은 간간 BDs3의빈약성을 초래한다. 또한, 다수의 급성 및 만성 간 질환은 담즙 마커를 발현하지만 담즙 세포 또는 형태에서 반드시 발생하지 않는 상당수의 세포의 존재로 정의되는 간에서 소위 연성 반응을 초래합니다. 특허 BDs4. 다중시스템 장애인 알라길증후군(ALGS)에서, 노치 리간드의 하플로인스실핍증1(JAG1)은BD 형성이 불량하고 담즙정체증5,6. 우리의 실험실은 최근에 이전에 생성 된 Jag1 이형 마우스 라인 7이 ALGS 8에서BD 빈곤의 동물 모델임을 입증했습니다. ALGS의 이 마우스 모델에서는 담관옥시트가 여전히 존재합니다. 그러나, 그들은 성숙한 특허 BDs 8에통합을 커밋하지 못합니다. 따라서 BD 빈곤의 모델에서 간을 분석하려면 담관옥시의 명백한 존재 또는 부재 이상이 필요합니다. 성숙한 BDs가 간에서 존재하는 정도를 정확하게 평가하는 것이 중요합니다.
해부학 병리학에서는 BD 빈곤이 존재하는지 여부를 평가하기위한 허용 된 양적 방법이있습니다 9. 예를 들어, 인간 환자에서 ALGS에 대한 연구는 간 생검9,10개당 적어도 10개의 포탈 혈관을 분석하여 BD를 포탈 정맥(PV) 비율로 정량화한다. 혈청 화학과 결합된 특허 BD의 모양 및 전반적인 존재 또는 부재의 분석은 마우스11,12,13에서BD 개발에 대한 귀중한 정보를 제공할 수 있다. 그러나, 마우스는 혈청 빌리루빈 수준 8에 있는 단지 겸손한 증가와BDs의 상당한 수를 분실할 수 있습니다. 따라서, PV당 존재하는 BDs의 수를 평가하는 정량적 방법은 마우스에서 BD 빈곤의 정도를 보다 직접적으로 측정할 수 있다. 최근 보고서에서, 우리는 모든 간 엽에 걸쳐 PV 당 BDs의 수를 정량화하고 Jag1 +/-- 동물8에서PV 비율에 BD에 있는 중요한 감소를 보고했습니다. 분석 과정에서 염증 반응 및 연성 반응의 정도가 현저한 변동에도 불구하고 BD 대 PV 비율은 많은가변성을 나타내지 않는 것으로 나타났습니다 8. 더욱이, BD 대 PV 비율의 정량화는 우리가 Jag1+/-에서 글리코실트랜스퍼라제 유전자 Poglut1의 1개의 사본을제거하는 것이 그들의 BD 빈곤8을 현저하게 향상시킬 수 있다는 것을 입증하는 것을 허용했다. Jag1+/+ 배경에서 혈관 평활근 세포에서 Poglut1의 조건부 손실은 BD 수치의 점진적 인 증가를 초래합니다(20-30%). P7에서 하지만 성인에서 눈에 띄는 된다8. 다시 말하지만, 이 기술은 P7에서도 이러한 동물의 BD 밀도의 증가가 통계적으로 유의하다는 것을 보여줄 수 있었습니다. 참고로, 생후 4개월에 이 유전자형의 증가된 BD 밀도는 수지 주조 분석을 통해서도 검증되었다. 8 다른 ALGS 마우스 모델14,15에서 BD 밀도를 측정한 이러한 관찰 및 기타 보고서는 다양한 돌연변이체의 담즙 결함을 분석하기 위한 전체 전략에 이 방법을 통합하도록 유도했습니다. 및 형질전환 마우스.
여기서, 간 질환의 마우스 모델에서 BD 빈곤의 정도를 검사하는데 사용될 수있는 간단한 기술을 상세히 기술한다(도 1). 이 방법에서, 콜란지오시포르테 마커 시토케라틴(CK) 8 및 CK19(이하 광스펙트럼 CK, wsCK)와 함께 공동 염색하는 것은 마우스 간에서 BD및 통합되지 않은 담관옥을 가시화하는데 사용된다. 알파 평활근 액틴(αSMA)에 대한 항체가 라벨 용기에 염색에 첨가된다. 모든 간 엽을 덮는 단면도에서 BD 대 PV 비율의 체계적인 분석은 각 유전자형에 대해 많은 수의 PV를 분석할 수 있도록 합니다. 우리의 방법은 2D 이미지에서 BD와 PV를 정량화에 의존하기 때문에, 담즙 나무의 3D 구조 또는 작은 담즙 도관의 무결성에 주어진 돌연변이의 효과를 연구하기에 적합하지 않습니다. 그럼에도 불구하고, 그것은 마우스에 담즙 발달을 평가하기 위하여 조사자가 간단하고 객관적인 전략을 제공합니다.

<table>
	<tbody>
		<tr>
			<td>Isothesia (Isoflurane)</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Bel-Art</td>
			<td>16-800-552</td>
			<td></td>
		</tr>
		<tr>
			<td>10% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15712</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tube</td>
			<td>ThermoScientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2401</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoChoice</td>
			<td>VWR Life Sciences</td>
			<td>H103-4L</td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>Omnisette Tissue Cassette</td>
			<td>Fisher HealthCare</td>
			<td>15-197-710E</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosette</td>
			<td>Simport</td>
			<td>M512</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>McCormick Scientific</td>
			<td>39503002</td>
			<td>Parrafin</td>
		</tr>
		<tr>
			<td>Tissue Mold</td>
			<td>Fisher Scientific</td>
			<td>62528-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM 325</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>C8H10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Based Antigen Retrieval</td>
			<td>Vector Laboratories</td>
			<td>H-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Cooker</td>
			<td>Instant Pot</td>
			<td>Lux Mini</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Pap Pen</td>
			<td>Life Technologies</td>
			<td>8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T. Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Octyl Phenol Ethoxylate (Triton-X-100)</td>
			<td>J.T. Baker</td>
			<td>X198-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CK8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>Antibody Registry ID AB531826</td>
		</tr>
		<tr>
			<td>anti-CK19</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-III</td>
			<td>Antibody Registry ID AB2133570</td>
		</tr>
		<tr>
			<td>anti-&#945;SMA</td>
			<td>Sigma Aldrich</td>
			<td>A2547, Clone 1A4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat-Alexa488</td>
			<td>ThermoFisher</td>
			<td>A21208</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse-Cy5</td>
			<td>Jackson Immunoresearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>22x50 micro cover glass</td>
			<td>VWR Life Sciences</td>
			<td>48393 059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech Science</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Antifade Mounting Medium</td>
		</tr>
	</tbody>
</table>

determining bile duct density in the mouse liver

마우스는 담즙 질병을 연구하는 모형 유기체로 광범위하게 이용됩니다. 담즙 계통의 발달 그리고 기능을 평가하기 위하여는, 특정 마커를 위한 혈청 화학, 조직학 분석 및 면역 얼룩을 포함하여 각종 기술이 이용됩니다. 이 기술은 담즙 시스템에 관하여 중요한 정보를 제공할 수 있더라도, 그(것)들은 수시로 전체 간을 통해 담관 (BD) 발달 결함의 전체 그림을 제시하지 않습니다. 이것은 부분적으로 담 즙 개발에 상당한 장애를 가진 동물에서 담 즙을 배출 하는 마우스 간의 강력한 능력 때문. 여기에서 우리는 돌연변이/형질전환 마우스의 모든 로브를 포함하는 단면도에 있는 각 문맥 (PV)와 관련되었던 BDs의 평균 수를 계산하는 간단한 방법을 제시합니다. 이 방법에서는, 간은 각종 유전형 및 실험 조건 사이 비교를 용이하게 하기 위하여 입체적인 방식으로 거치되고 단면화됩니다. BDs는 세포각증 염색 담관옥시의 가벼운 현미경 검사를 통해 확인된 다음 간 절편에 존재하는 총 PV 수로 계산및 분할됩니다. 예를 들어, 우리는이 방법이 명확하게 야생 형 마우스와 Alagille 증후군의 마우스 모델을 구별 할 수있는 방법을 보여줍니다. 여기에 제시된 방법은 담즙 나무의 3 차원 구조를 시각화하는 기술을 대체 할 수 없습니다. 그러나, BD 발달 및 마우스에 있는 연성 반응 대형의 정도를 정량적으로 평가하는 쉽고 직접적인 방법을 제공합니다.

우리는 이전에 Jag1 +/-동물, ALGS8의마우스 모델에서 담즙 결함을 문서화했습니다. BD 대 PV 비율을 결정하기 위해, 우리는 P30 마우스 간을 절제하고 혈관 마커 αSMA와 함께 CK8 및 CK19 (wsCK)를 위해 그들을 공동 염색했습니다. 그런 다음 각 간 엽의 모든 PV를 이미지화했습니다. 그림 2A에나타난 바와 같이, 우리는 인접한 wsCK 염색 (화...

Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

Determining Bile Duct Density in the Mouse Liver

우리는 마우스 간에서 담관 밀도의 정확한 정량화를 위한 다소 간단하고 민감한 방법을 제시합니다. 이 방법은 유전 및 환경 수정자의 효과와 담즙 질환의 마우스 모델에서 잠재적 인 치료의 효과를 결정하는 데 도움이 될 수 있습니다.

마우스 간에서 담관 밀도 결정

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

이 프로토콜은 조사자가 간 질환의 마우스 모델에서 담관 개발을 정량화 할 수 있게합니다. 그것은 또한 담관 개발에 유전 수정자의 효과 평가에 대 한 허용. 이 기술의 장점은 담관 개발의 직접 평가를 위한 간단하고 민감하며 정량적인 방법이라는 것입니다.먼저, 포셉에 의해 팽팽하게 피부가 고정되어 있는 가운데, 작은 가위를 사용하여 안락사 된 마우스의 갈비뼈 아래 약 1 인치 의 횡절개를 합니다. 간 의 전체 복부 표면을 노출. 작은 가위를 사용하여 복부에 있는 그밖 기관에 간을 연결하는 인대를 주의 깊게 잘라냅니다.그런 다음 일반적인 담관을 잘라 내어 장에서 간을 분리합니다. 담낭을 잡고 간을 조심스럽게 제거하십시오. 즉시 4 %의 파라 포름알데히드로 3 분기 가득 50 밀리리터 튜브에 배치합니다.간 조직을 해결하려면 48 시간 동안 섭씨 4도에서 4 %의 PFA로 배양하십시오. 일련의 에탄올 세척 후, 실온에서 각각 30 분 동안 클리어링 에이전트로 간을 세 번 씻으릅니다. 포함을 시작하려면, 60섭씨까지 예열된 파라핀 왁스에서 30분 동안 티슈 몰드에 티슈 카세트를 세 번 씻는다.그런 다음, 3 분기 높이에 파라핀 왁스로 조직 금형을 채우고 섭씨 60도에서 가열 블록에 보관하십시오. 복부 쪽이 위로 향하여 금형에 간을 놓습니다. 가열 블록에서 금형을 조심스럽게 제거 한 후, 금형에 카세트의 상단을 배치합니다.뜨거운 액체 파라핀으로 위로 얹고 밤새 실온으로 식힙니다. 곰팡이에서 간 블록을 제거 한 후, 미세 토메를 사용하여 간 표면 등쪽을 통해 단면화하여 5 마이크로 미터 섹션을 만듭니다. 해부 현미경의 밑에 피상적 인 단면을 확인하여 섹션이 전단되거나 접히지 않았는지 확인하십시오.면역히스토케작용을 위한 슬라이드를 처리하려면 유전자형당 슬라이드 1개를 선택하고 자일렌, 100% 에탄올, 95%에탄올, 마지막으로 70%에탄올로 분석하여 15분 동안 세척한다. 이온화 된 물에 슬라이드를 세척 한 후, 트리스 기반의 높은 pH 항원 검색 용액에 담급. 그런 다음 평방 인치 당 10 파운드에서 3 분 동안 압력 밥솥에 압력을 가하여 가열하십시오.슬라이드를 실온으로 식힌 후 PAP 펜을 사용하여 슬라이드의 섹션을 간략하게 설명합니다. PBS Tween으로 세척한 후, 섹션당 블로킹 용액 100마이크로리터를 추가하고 1시간 동안 섭씨 4도에서 배양하여 조직 섹션을 차단합니다. 각 섹션에 3개의 1차 항체를 모두 포함하는 희석된 항체 용액의 100마이크로리터를 적용하고 하룻밤 사이에 섭씨 4도에서 배양한다.슬라이드를 세척한 후, 실온에서 1시간 동안 이차 항체와 인큐베이터를 모두 포함하는 이차 항체 용액의 100마이크로리터를 추가한다. 마지막으로 슬라이드에 마운팅 미디엄과 커버슬립을 적용합니다. 형광 현미경을 사용하여 각 섹션의 1x 확대/축소에서 20배 이미지를 촬영합니다.간을 가로지르는 모든 포탈 정맥이 이미지화되었는지 확인합니다. 다음 열이 있는 스프레드시트를 만듭니다: 동물/샘플 번호, 이미지 번호, 포털 정맥 수 및 담즙 덕트 수입니다. 각 이미지의 이미지당 포털 정맥 수를 식별하고 기록합니다.그런 다음, 정의 가능한 루멘을 둘러싼 담랑고이트의 존재에 의해 각 이미지에서 특허 담관을 식별합니다. 이 구조물은 그밖 광스펙트럼 사이토케라틴 양성 세포에서 mesenchyme에 의해 구별되고 분리되어야 합니다. 이 기술의 한 가지 주요 과제는 특허 담관을 식별하는 것입니다.특허 덕트란 무엇을 결정하는 것은 덕트의 모양과 루멘을 둘러싼 세포의 분석을 필요로 한다. 각 특허 담관을 계산하고 이미지 번호와 동일한 열에 배치하고 각 포털 정맥 이미지에 대해 반복합니다. 마지막으로, 간 샘플의 모든 포탈 정맥 및 모든 담관의 합을 계산하고 간 샘플에 대한 담관비율을 포문 정맥 비율로 계산합니다.P30 마우스 간은 혈관 마커, 알파-SMA와 함께 CK8 및 CK19를 위해 단면화 및 공동 염색되어 BD 대 PV 비율을 결정하였다. 각 간 엽의 모든 포탈 정맥이 이미지화되면, 포탈 정맥은 인접한 광스펙트럼 사이토케라틴 염색이 있는 알파-SMA 염색 혈관으로 정의되었다. 광범위한 사이토케라틴이 없는 알파-SMA 얼룩 구조는 분석에서 제외된 중앙 정맥이었다.특허 덕트는 광스펙트럼 시토케라틴 양성 담랑기옥예스로 둘러싸인 명백히 정의 가능한 루멘을 가지고 있으며, 일반적으로 인근 덕트 또는 담랑고이테에서 메센키메로 분리된다. 정의 가능한 루멘이없는 넓은 스펙트럼 사이토케라틴 양성 세포는 담관의 총 수로 계산되지 않습니다. 야생형 동물의 간 단면은 여러 개의 비법인 세포와 함께 완전히 특허 덕트와 관련된 포문 정맥을 나타낸다.JAG1 이종수 동물의 대표적인 간부는 3개의 포탈 정맥 주변에 특허 담관이 존재하지 않는다는 것을 보여줍니다. 여기에 모든 넓은 스펙트럼 사이토케라틴 양성 세포는 통합되지 않으므로 계산해서는 안됩니다. 3개의 야생형 및 3개의 JAG1 이종수구동물에 대한 BD 대 PV 비율을 분석한 결과 담관 수에 기초하여 두 가지 유전자형을 쉽게 구별할 수 있는 방법을 보여줍니다.담관에 대한 모든 포털 선박을 평가하는 것이 중요합니다. 그것은 간을 통해 표형 가변성이 있는 경우에 담관 밀도를 결정하기위한 특히 중요합니다. 이 기술은 헬가슨의 마우스 모형에 있는 담즙 표현형의 유전 억제기를 확인하기 위하여 이용되었습니다.따라서, 담즙 질환의 동물 모델의 치료 양식에 유용할 수 있다.

determining-bile-duct-density-in-the-mouse-liver

Research

JoVE Journal

Developmental Biology

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

마우스 파골 세포 전구체의 노치 신호 전달의 자극

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

연구

발생학

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Zebrafish에서의 급성 알코올성 간 손상의 조직 학적 분석

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

생체 외에서 마우스 시뮬레이션된 Microgravity에서 Preantral 모 낭의 성장

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

Sholl 분석법을 이용한 유방 내 유선 마운트의 분지 밀도 정량화

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

마우스에서 초기 임신 특성에 대 한 3 차원 높은-주파수 초음파의 새로운 사용

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

가족성 콜레스테롤 혈 증 인간의 간 공상 마우스 모델을 사용 하 여 유도 만능 줄기 세포 유래 Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Organoids 마우스 Extrahepatic 담 즙 덕트에서의 세대

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...