Name: three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary
Uploaded: 2017-12-10T14:00:00.000+00:00
Duration: 12 min 38 s
Description: Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

이 연구는 초나라에 대 한 중국 특별 기금에서 교부 금에 의해 지원 되었다 (YF No. 2014T70392), 국립 자연 과학 재단의 중국 YF (No. 81673766), 새로운 교사를 못쓰게 기금, 복 단 대학의 Zuoxue 재단 및 개발 상하이 최대 분야 통합 의학 (20150407)의 프로젝트.

동물 주제와 관련 된 모든 절차에서 상해 의학 대학, 복 단 대학 (승인 번호 20160225-013) 동물 윤리 위원회의 지침에 따 랐 다.
1입니다. 투명 마우스 난소의 준비
<ol><li>
솔루션의 준비
	<ol>
<li>준비 인산 염 버퍼 식 염 수 (PBS) 솔루션 (1 M, pH 7.6) 0.1% 트라이 톤 X-100 (PBST). 10 x PBS 재고 솔루션 1 L을 혼합 87 g의 NaCl, NaH2포4 · 3.1 g 2 H2O, 그리고 나2HPO4 ·12H2오의 28.7 g dH2O L 1과 볶음 ~ 2 h. pH 7.4에 조정 1 N NaOH를 추가 하 고 상 온에서 저장. 1/10이 10 배 재고 솔루션을 희석 0.1% 이며 dH2O를 사용 하 여 PBS에 트라이 톤 X-100.</li>
		<li>Mixings 4% (wt/vol) paraformaldehyde (PFA) 솔루션, 4% (wt/vol) 아크릴, 0.05% (wt/vol) bisacrylamide, 및 0.25% (wt/vol) VA-044 초기자 두 번 증 류 물 얼음에 의해 히드로 솔루션 (pH 7.5)를 준비 합니다.</li>
		<li>4% (wt/vol) 나트륨 라우릴 황산 염 (SDS) 이중 증 류 물에서 포함 된 200 m m 나트륨 붕 산 염 버퍼를 혼합 하 여 개간 솔루션 (pH 8.5)를 준비 합니다.</li>
	</ol>
</li>
	<li>
Transcardial 관류와 난소 준비
	<ol>
<li>모든 절차 동안 얼음에 모든 솔루션을 유지.</li>
		<li>성인 여성 와일드 타입 C57BL/6 마우스 pentobarbital (0.35 mg/kg) 솔루션의 복 주사와 anesthetize 발가락-핀치 응답 보여줍니다, 일단 마우스 제대로 마 취.</li>
		<li>사지와 마우스를 해결 하기 위해 사용 스티커 테이프 플랫 폼 보드에 밖으로 뻗어.</li>
		<li>가 위로 가슴, 피부, 뼈를 통해 칼 프로세스에 절 개를 마음에 노출.</li>
		<li>동물의 오른쪽 아 트리 움로 절 개 하 고 perfuse 10 mL/min에서 얼음 1 x PBS의 20 mL와 좌 심 실.</li>
		<li>20 mL 이상 10 mL/min에서 히드로 솔루션으로 동물 perfuse</li>
		<li>가 위, 전체 enterocoelia를 노출, medioventral 라인을 따라 복 부에 피부 incise 고 내장을 제거 합니다. 난소 뿐만 아니라 자 궁 난소 형태학의 무결성을 유지 하기 위해 수집 합니다.</li>
		<li>현미경으로 난소 주위 지방 조직을 제거 합니다.</li>
	</ol>
</li>
	<li>
기체 제거
	<ol>
<li>3 일 동안 고정을 위한 4 ° C에서 10 mL 히드로 솔루션에 난소를 놓습니다.</li>
		<li>5 mL 관으로 난소를 전송 하 고 히드로 솔루션 튜브를 입력 합니다. 튜브 위에 parafilm 스트레칭 하 고 튜브의 목 parafilm 포장. 주의:는 parafilm와 히드로 솔루션 사이 아무 거품 인지 확인 합니다.</li>
		<li>유해를 하이드로 겔 수 있도록 3 h의 최대 37 ° C에서 인큐베이터에서 통에 튜브를 놓습니다.</li>
		<li>주걱을 사용 하 여 젤에서 난소를 제거 하 고 조심 스럽게 티슈 페이퍼에 의해 난소 주위에서 초과 젤을 제거. 4 번 1 x PBS 가진 난소를 씻어.</li>
	</ol>
</li>
	<li>
수동 청소
	<ol>
<li>청소 솔루션의 10 mL에 난소 잠수함 개간 과정을 시작 하는 37 ° C에서 80 rpm에서 부드럽게 흔들어.</li>
		<li>첫 주에 3 일 마다 솔루션을 변경 합니다. 일단 난소까지 주 분명해 1 주일 후 청소 솔루션을 새로 고칩니다. 일반적으로이 프로세스는 약 2-3 주 걸립니다. 참고: 난소 또는 immunostained를 직접 수 저장할 수 있습니다 나트륨 아 지 드 (0.02%)에 PBS에 4 ° C에서 immunostaining 전에 몇 주 동안.</li>
	</ol>
</li>
</ol>2입니다. Immunostaining
<ol><li>통에 37 ° C에서 1 일에 PBST에 두 번 난소를 씻어.</li>
	<li>37 ° c.에 2-3 일에 1 차 항 체/PBST 솔루션 (표 1)에 통에 난소를 품 어 에 여기에 제시, 기본 항 체 혈소판 내 피 성장 세포 접착 분자 1 (CD31) 했다 일과 티로신 hydroxylase 각각 1시 10분, PBST에 1시 50분에 희석. 주의: 두 개 이상의 종류 1 차적인 항 체의 사용 하는 경우 다른 동물 소스 있는지 확인 합니다.</li>
	<li>하루에 4 통에 37 ° C에서 PBST 버퍼 기본 항 체를 씻어.</li>
	<li>하루 5-6 통에 37 ° C에 PBST에서 원하는 보조 항 체와 난소를 품 어. 여기에 제시 된 작품에 2 차 항 체 티로신 hydroxylase와 알 렉 사 가루 594 CD31 PBST에 1시 50분에 희석에 대 한 알 렉 사 가루 488을 했다.</li>
	<li>통에 7 일에 37 ° C에서 PBST 버퍼와 2 차 항 체를 씻어.</li>
	<li>주 8에 굴절률 균질에 대 한 굴절률 매칭 솔루션으로 조직의 전송. 눈금선의 시각적 선명도 눈금선을 볼 수 있는지 여부를 확인 하 여 확인 하는 종이에 조직을 넣어. 일단 조직을 완전히 명확히 있다, 몇 군데 될 수 있습니다.</li>
	<li>단지 주변을 수 퍼 실린더 조직, 그리고 말굽 모양으로 모양을 만들고 밀폐 공간을 만들기 위하여 유리 슬라이드에 밀접 하 게 외부 릿지를 누릅니다. 퍼 티의 높이 난소 보다 조금 키가 있어야 합니다.</li>
	<li>명확히 난소 신중 하 게 퍼 티의 중심에 놓고 굴절률 일치 하는 솔루션을 추가 합니다. 유리 하단 접시 퍼 티에 단단히 넣고 퍼 티를 사용 하 여 요리를 해결.</li>
</ol>3. confocal 현미경 검사 법
<ol><li>Confocal 현미경 이미징, "Ni-E" 패널에서 선택 (이 경우 물 침수 25 X 렌즈, 1.1-나, 2 mm-WD)에 렌즈 (이 경우 2.0 m m)에 적절 한 작업 거리.</li>
	<li>
"수집" 패널에서 채널 수를 선택 합니다. 여기에, 3 개의 채널을 사용 합니다.
	<ol>
<li>첫째, 사용 하 여 "눈 포트" 탭 및 XY 컨트롤러 조이스틱 현미경 접 안 렌즈를 통해 확인 하 여 조직 필드의 중심을 이동.</li>
		<li>셔터 라이트를 끈 후 조정 "레이저 파워", "HV (이득)", "라이브" 탭을 클릭 하 고 "오프셋 ' 각 별도로 채널. 높은 레이저 전원 및 HV 높은 신호를 발생할 수 있습니다 그리고 더 높은 오프셋 배경 잡음을 줄일 수 있습니다.</li>
		<li>적절 한 "스캔 크기"와 "수"를 선택 합니다. 사용 하 여 1024 x 1024 µ m2 의 스캔 크기와 4 ~ 8의 수 있습니다.</li>
	</ol>
</li>
	<li>
설정 패널에서 조직의 깊이 정의 하기 위한 이미지 품질 "Z 강도 보정", 후 (조직 중심)에서 난소의 최상위 계층에 렌즈를 찾아서 최상위 계층에 대 한 인수를 조정 후, 클릭 "더하기" 버튼을 상위 계층을 정의 합니다.
	<ol>
<li>렌즈를 이동 하 고 적절 한 HV 및 조직의 하단에 대 한 레이저 전원 설정 Z 강도 보정 테이블에 낮은 레이어 정보 추가. "하 차" "ND 수집" 패널 설정을 동기화 하려면 클릭 합니다.</li>
	</ol>
</li>
	<li>
"ND 수집" 패널, 스캔 수 단계 첫 번째 집합. 그런 다음, "Z"와 "큰 이미지" 탭 체크.
	<ol>
<li>접 안경 통해 조직 시각화의 도움으로 조직의 경계에 따라 필드의 크기를 정의 하려면 "스캔 큰 이미지" 창으로 이동 합니다. 다시 검색 영역 ("필드")의 크기를 입력 하 고 10%와 15% 사이 오버랩 선택 "ND 수집" 패널으로 이동.</li>
		<li>"실행 Z 수정" 하지 "실행" 지금", 자동으로 각 계층에 대 한 적절 한 HV, 레이저 파워와 오프셋을 설정 하 고 이미지 처리를 위해 대기를 클릭 합니다.</li>
	</ol>
</li>
</ol>4. 개별 낭과 그들의 Vasculatures의 3D 재구성
<ol><li>첫째, ImageJ5 를 사용 하 여 원래 NIS 파일을 엽니다. "이미지" 버튼을 클릭 하 고 "색상" 및 "분할 채널"을 선택 합니다. 다음, "파일"을 클릭 하 고 별도로 "다른 이름으로 저장"에서 "이미지 시퀀스" 옵션을 사용 하 여 tiff 파일 시리즈로 채널을 저장.</li>
	<li>Imaris를 사용 하 여 tiff 파일 시리즈 중 하나를 엽니다. "편집" 버튼을 클릭 하 고 "채널 추가"를 선택 추가 채널의 나머지를. 이름 및 색상 채널의 "디스플레이 조정" 탭에서 변경할 수 있습니다. 직물의 두께 "편집" 드롭 다운 목록에서 "이미지 속성" 패널에서 수정 해야 합니다.</li>
	<li>3D 자르기 최종 이미지의 과잉 부분 제거, 대 한 "편집" 버튼을 클릭 하 고 "3D 자르기"를 선택 합니다. 테두리를 끌어 필드의 크기를 조정할 수 있습니다.</li>
	<li>
Surpass 패널에 필 라 멘 트 알고리즘으로 선박 신호를 재구성 하 새로운 필 라 멘 트를 생성 하 고 클릭 합니다 "필" 버튼을 클릭 합니다.
	<ol>
<li>가장 크고 가장 얇은 직경을 정의 하기 위해 "슬라이스" 패널에가 고 추적 선박을 찾을. 거리 선박의 최대 너비에 두 포인트를 선택 하 여 "측정" 패널에 자동으로 표시 됩니다.</li>
		<li>"Surpass" 패널에 돌아가 측정된 폭을 입력을 클릭 합니다.</li>
	</ol>
</li>
	<li>시작 및 씨앗 포인트 임계값을 조정 합니다. "카메라" 패널에서 "선택"을 선택 합니다. 자동으로 생산된 분야의 일부는 제거 될 필요가, shift 하 고 지점에 클릭 하십시오. "이동"을 선택 하 고 마우스를 이동, 구조는 모든 올바른 포인트 유지 되 고 다음을 클릭 합니다 회전할 수 있습니다.</li>
	<li>로컬 대비를 위한 높은 임계값을 선택 하 고을 클릭 합니다. "쪽이 검출"를 선택한 혈관 실린더의 재건을 마무리 계속 하지 마십시오. 색상은 "색상" 탭을 클릭 하 여 편집할 수 있습니다. 통계 데이터는 복원 된 필 라 멘 트의 "통계" 패널에서 추출할 수 있습니다. "편집" 패널에서 초과 부분을 제거할 수 있습니다. 참고: 다른 알고리즘은 총 낭 또는 소 낭 모양 벽의 볼륨을 측정 하는 데 사용할 수 있습니다. 여기에 제시 된 작품에는 oocytes 두 이차 항 체의 autofluorescence에 의해 표를 했다. 많은 양의 데이터 때문에 워크스테이션 서버 데이터 분석을 위해 사용 되었다.</li>
</ol>5. 통계 분석
참고: 이미지 분석 데이터는 ± 표준 오류를 의미 하는 대로 표시 됩니다.
<ol><li>독립 표본 t-검정를 사용 하 여 모 공 사이의 비교를 수행 하 고 피어슨의 상관 관계 테스트를 사용 하 여 상관 관계 계수 follicular 벽 및 모 세관 길이 비교. P의 값을 설정 < 0.05 통계적 의미에 대 한 제한.</li>
</ol>

<ol>
	<li>Brown, H. M., Russell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+and+lymphatic+vasculature+in+the+ovary:+development,+function+and+disease.">Blood and lymphatic vasculature in the ovary: development, function and disease.</a> Hum Reprod Update. 20 (1), 29-39 (2014).</li><li>McFee, R. 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=Inhibition+of+vascular+endothelial+growth+factor+receptor+signal+transduction+blocks+follicle+progression+but+does+not+necessarily+disrupt+vascular+development+in+perinatal+rat+ovaries.">Inhibition of vascular endothelial growth factor receptor signal transduction blocks follicle progression but does not necessarily disrupt vascular development in perinatal rat ovaries.</a> Biol Reprod. 81 (5), 966-977 (2009).</li><li>Coveney, D., Cool, J., Oliver, T., Capel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-dimensional+analysis+of+vascularization+during+primary+development+of+an+organ,+the+gonad.">Four-dimensional analysis of vascularization during primary development of an organ, the gonad.</a> Proc Natl Acad Sci U S A. 105 (20), 7212-7217 (2008).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nat Protoc. 9 (7), 1682-1697 (2014).</li><li>Schindelin, J., 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=Fiji:+an+Open+Source+platform+for+biological+image+analysis.">Fiji: an Open Source platform for biological image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Cao, G., Fehrenbach, M. L., Williams, J. T., Finklestein, J. M., Zhu, J. X., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+platelet+endothelial+cell+adhesion+molecule-1-null+mice.">Angiogenesis in platelet endothelial cell adhesion molecule-1-null mice.</a> Am J Pathol. 175 (2), 903-915 (2009).</li><li>Manni, L., Holm&#228;ng, A., Lundeberg, T., Aloe, L., Stener-Victorin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+expression+of+alpha+(1)-+and+beta+(2)-adrenoceptors+and+p75+neurotrophin+receptors+in+rats+with+steroid-induced+polycystic+ovaries.">Ovarian expression of alpha (1)- and beta (2)-adrenoceptors and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries.</a> Auton Neurosci. 118 (1 - 2), 79-87 (2005).</li><li>Chourasia, T. K., Chaube, R., Singh, V., Joy, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annual+and+periovulatory+changes+in+tyrosine+hydroxylase+activity+in+the+ovary+of+the+catfish+Heteropneustes+fossilis.">Annual and periovulatory changes in tyrosine hydroxylase activity in the ovary of the catfish Heteropneustes fossilis.</a> Gen Comp Endocrinol. 166 (1), 111-116 (2010).</li><li>Feng, Y., 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=CLARITY+reveals+dynamics+of+ovarian+follicular+architecture+and+vasculature+in+three-dimensions.">CLARITY reveals dynamics of ovarian follicular architecture and vasculature in three-dimensions.</a> Sci Rep. 7, 44810 (2017).</li><li>Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+principles+in+tissue+clearing+and+staining+protocols+for+whole-body+cell+profiling.">Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.</a> Annu Rev Cell Dev Biol. 32, 713-741 (2016).</li><li>Liang, H., Schofield, E., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Serotonergic+Fibers+in+the+Mouse+Spinal+Cord+Using+the+CLARITY/CUBIC+Technique.">Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique.</a> J Vis Exp. (108), e53673 (2016).</li><li>Yang, B., 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=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li><li>Phillips, J., Laude, A., Lightowlers, R., Morris, C. M., Turnbull, D. M., Lax, N. Z. <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+passive+CLARITY+and+immunofluorescent+labelling+of+multiple+proteins+in+human+cerebellum:+understanding+mechanisms+of+neurodegeneration+in+mitochondrial+disease.">Development of passive CLARITY and immunofluorescent labelling of multiple proteins in human cerebellum: understanding mechanisms of neurodegeneration in mitochondrial disease.</a> Sci Rep. 6, 26013 (2016).</li><li>Roberts, D. G., Johnsonbaugh, H. B., Spence, R. D., MacKenzie-Graham, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+clearing+of+the+mouse+central+nervous+system+using+passive+CLARITY.">Optical clearing of the mouse central nervous system using passive CLARITY.</a> J Vis Exp. (112), (2016).</li><li>Woo, J., Lee, M., Seo, J. M., Park, H. S., Cho, Y. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+optical+transparency+of+rodent+tissues+by+modified+PACT-based+passive+clearing.">Optimization of the optical transparency of rodent tissues by modified PACT-based passive clearing.</a> Exp Mol Med. 48 (12), e274 (2016).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li><li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annu Rev Cell Dev Biol. 27, 563-584 (2011).</li><li>Rodgers, R. J., Irving-Rodgers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+ovarian+follicular+antrum+and+follicular+fluid.">Formation of the ovarian follicular antrum and follicular fluid.</a> Biol Reprod. 82 (6), 1021-1029 (2010).</li><li>Siu, M. K. Y., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-follicle+barrier+(BFB)+in+disease+and+in+ovarian+function.">The blood-follicle barrier (BFB) in disease and in ovarian function.</a> Adv Exp Med Biol. 763, 186-192 (2014).</li><li>Dodt, H. U., 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=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat Methods. 4, 331-336 (2007).</li><li>Hama, H., 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=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nat Neurosci. 14, 1481-1488 (2011).</li><li>Erturk, 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nat Protoc. 7, 1983-1995 (2012).</li><li>Kuwajima, T., 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=Clear(T):+a+detergent-+and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">Clear(T): a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140, 1364-1368 (2013).</li><li>Ke, M. T., Fujimoto, S., Imai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeeDB:+a+simple+and+morphology-preserving+optical+clearing+agent+for+neuronal+circuit+reconstruction.">SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.</a> Nat Neurosci. 16, 1154-1161 (2013).</li><li>Lai, H. 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=Rationalisation+and+validation+of+an+acrylamide-free+procedure+in+three-dimensional+histological+imaging.">Rationalisation and validation of an acrylamide-free procedure in three-dimensional histological imaging.</a> PLOS ONE. 11, e0158628 (2016).</li><li>Susaki, E. 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157, 726-739 (2014).</li><li>Tainaka, K., 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=Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization.">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159, 911-924 (2014).</li><li>Liu, A. K. L., Lai, H. M., Chang, R. C. C., Gentleman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-of-acrylamide+SDS-based+tissue+clearing+(FASTClear):+A+novel+protocol+of+tissue+clearing+for+three-dimensional+visualisation+of+human+brain+tissues.">Free-of-acrylamide SDS-based tissue clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.</a> Neuropathol Appl Neurobiol. 43, 346-351 (2016).</li><li>Xu, 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=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+three-dimensional+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.</a> Sci Rep. 7, 9895 (2017).</li><li>Migone, F. F., Cowan, R. G., Williams, R. M., Gorse, K. J., Zipfel, W. R., Quirk, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+an+essential+role+of+vasoconstriction+in+rupture+of+the+ovarian+follicle+at+ovulation.">In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.</a> Proc Natl Acad Sci U S A. 113, 2294-2299 (2016).</li><li>Malki, S., Tharp, M. E., Bortvin, 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+whole-mount+approach+for+accurate+quantitative+and+spatial+assessment+of+fetal+oocyte+dynamics+in+mice.">A whole-mount approach for accurate quantitative and spatial assessment of fetal oocyte dynamics in mice.</a> Biol Reprod. 93 (113), (2015).</li></ol>

저자는 공개 없다.

현재 연구에서 선물이 3D 이미징 모세 혈관과 개별 성장 낭 사이의 관계를 평가 하기 위해. 우리의 이전 작품에서는 동일한 프로토콜 9를 사용 하 여, 우리는 큰 맥 관 구조, 모 공, 그대로 마우스 난소에서 난 포의 위치 사이 상호 작용의 역할을 공부 했습니다. 수동 선명도 접근 수 마이크로 및 매크로 vasculatures, folliculogenesis, corpora lutea의 다른 발달 단계에서 난소 아키텍처를 재구...

여 포는 난소의 기본적인 구조상과 기능 단위 이며 그 개발 높은 난소 내에서 맥 관 구조에 관련 된. 혈관 영양과 모 낭에 호르몬을 공급 하 고 따라서 성장과 낭1의 성숙에 중요 한 역할.
선택적 혈관 마커, 유전자 변형 마우스 모델 및 제약 개발을 포함 하 여 기술의 조합 난소 혈관 네트워크, 신생, 혈관의 기능에 대 한 우리의 지식을 증가합니다 folliculogenesis입니다. 난소는 그것 folliculogenesis와 배 란 기간 동안 다양 한 조직 및 혈관 네트워크를 개장 하기 때문에 활성 장기로 알려져 있다. 혈관의 구조와 크기에 이러한 활성 개장 하는 것은 개발 및 낭의 생물 학적 기능을 위한 필요 이다.
난소 및 혈관의 immunolabeling를 사용 하 여 전통적인 조직학 및 histomorphometric 메서드는 2 차원 (2D) 이미지2. 3 차원 (3D) 재구성 기술 개발 조직 조각의 2D 이미지 3 차원 구조를 만들기 위해 중첩 수 있습니다 하지만이 방법은 여전히 몇 가지 한계가-조직의 단면 마이크로 구조의 일부를 파괴할 수는 조직, 자주 그리고 상당한 노동은 조각에서 얻은 이미지에서 3 차원 개조를 제작에 관련 된. Confocal 현미경 검사 법으로 전체 조직의 3D 영상 많은, 이러한 한계를 극복할 수 있지만 이러한 방법은 배아 난소3신생의 평가에 제한 됩니다. 전체 조직의 선명도4 같은 방법의 선택을 취소를 사용 하 여 산 후 및 성인 난소에 이러한 문제를 해결할 수 있도록 시각화 된 볼륨을 증가 시킬 수 있습니다 그리고 이러한 방법을 모든 구조적 변형 없이 난소의 광학 정리 제공. 그대로 난소의 3D 아키텍처의 이미징이이 작품에 사용 된 Imaris 소프트웨어 패키지 같은 이미지 분석 소프트웨어에 대 한 정확한 이미지 데이터베이스를 제공 합니다.
성인 기에 걸쳐 난소의 개장 동적 생리 체계의 일부 이며 이것은 난소 신생의 규칙에 대 한 조사에 대 한 우수한 모델. 또한, polycystic 난소 증후군이 나 난소 암 등 여성 생식의 pathologic 상태에서 난소 혈관의 역할을 평가 전체 난소 조직 영상 통해 공부 될 수 있다. 수동 선명도 방법의 개발 및 고급 이미지 분석 소프트웨어를 사용 하 여 혈관과 모 낭 등 난소의 구조 간의 관계에 자세한 공간 정보 제공.

<table><tbody><tr><td>Acrylamide</td><td>Vetec</td><td>v900845</td><td>http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td></tr><tr><td>Alexa Flour 488 (Dilution 1:50)&amp;nbsp;</td><td>Life Technologies</td><td>A11039</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td></tr><tr><td>Alexa Flour 594 (Dilution 1:50)</td><td>Life Technologies</td><td>A11012</td><td>https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td></tr><tr><td>Bisacrylamide</td><td>Amresco</td><td>172</td><td>http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td></tr><tr><td>Black wall glass bottom dish (Willco-Dish)</td><td>Ted Pella</td><td>14032</td><td>http://www.tedpella.com/section_html/706dish.htm#black_wall</td></tr><tr><td>Boric acid</td><td>Sinopharm Chemical Reagent</td><td>10004818</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td></tr><tr><td>Disodium hydrogen phosphate dodecahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 12H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>10020318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td></tr><tr><td>FocusClear</td><td>Celexplorer</td><td>FC-102</td><td>http://www.celexplorer.com/product_list.asp?MainType=107&amp;amp;BRDarea=1</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td>http://www.parafilm.com/products</td></tr><tr><td>Paraformaldehyde</td><td>Sinopharm Chemical Reagent</td><td>80096618</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td></tr><tr><td>PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td><td>Abcam</td><td>ab28364</td><td>http://www.abcam.com/cd31-antibody-ab28364.html</td></tr><tr><td>Photoinitiator VA044</td><td>Wako</td><td>va-044/225-02111</td><td>http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td></tr><tr><td>Sodium azide</td><td>Sigma</td><td>S2002</td><td>http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&amp;amp;region=US</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sinopharm Chemical Reagent</td><td>10019318</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td></tr><tr><td>Sodium dihydrogen phosphate dihydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sinopharm Chemical Reagent</td><td>20040718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sinopharm Chemical Reagent</td><td>30166428</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sinopharm Chemical Reagent</td><td>10019718</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td></tr><tr><td>Triton X-100</td><td>Sinopharm Chemical Reagent</td><td>30188928</td><td>http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td></tr><tr><td>Tyrosine hydroxylase (TH, Dilution 1:50)</td><td>Abcam</td><td>ab76442</td><td>http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td></tr></tbody></table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

난소는 여성의 생식 시스템의 주요 기관 이다 하 고 여성 gametes의 생산에 대 한 복잡 한 구조 관계와 3 차원 (3D) 맥 관 구조 아키텍처의 내 분 비 시스템을 제어 하기 위한 필수적 이다는 난소는 잘 기술 된다. 3D 연결과 그대로 난소에 혈관의 아키텍처를 시각화 하기 위해 첫 번째 중요 한 단계는 광학 투명 난소를 만드는 것입니다. 피하 조직의 수축, 우리가 사용 하는 하이드로 겔 고정 기반 수동 선명도 (아크릴 때 교배 고정 취소 지질 교환 이미징 / Immunostaining/에서 제자리 교 잡-호환 조직 히드로) 메서드는 그대로 난소를 프로토콜 . Immunostaining, 고급 multiphoton confocal 현미경 검사 법, 그리고 3D 이미지 복원 다음 난소 혈관 및 소 낭 모양 모 세관의 시각화를 위해 사용 되었다. 이 방식을 사용 하 여, 우리는 상당한 긍정적인 상관 관계 보였다 (P < 0.01) follicular 벽의 볼륨과 follicular 모 세관의 길이 사이.

우리는 수동 난소 follicular와 혈관 구조를 보존 하 고 혈관과 모 낭의 레이블이 표시에서 높은 형광 신호를 취득 하는 동안 지우기에 대 한 신속 하 고 간단한 방법으로 수동 선명도 메서드를 적응. Follicular 맥 관 구조의 3D 아키텍처 CD31, 내 피 세포6표식에 대 한 immunostaining에 의해 결정 되었다. 필 라 멘 트 알고리즘을 사용 하 여 및 follicular 모 세관 및 기본...

Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

여기 우리는 그대로 마우스 난소에서 수동 선명도 난소 맥 관 구조 및 follicular 모세 혈관의 시각화를 위한 3D 재구성 방법의 적응을 제시.

수동 선명도 삭제 마우스 난소의 혈관 구조의 3 차원 재구성

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

three-dimensional-reconstruction-vascular-architecture-passive

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.6K 조회수.  Fudan University. 여기 우리는 그대로 마우스 난소에서 수동 선명도 난소 맥 관 구조 및 follicular 모세 혈관의 시각화를 위한 3D 재구성 방법의 적응을 제시.

비디오: Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

에 대한 면역 적격 마우스에 묽은 난소암의 Orthotopic 모델 생체내에</em종양 면역 응답> 종양 이미징 및 모니터링

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

연구

면역학 및 감염병학

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

3 차원 렌더링 및 Immunolabeled, 분석 인간 Placental Villous 혈관 네트워크를 명확히

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

발생학

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

전체 산 면역 형광 및 교양된 마우스 난소의 여 포 정량화

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

난소 조직 문화 현상을 마우스 난소에서 시각화 하

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

영상은 3DISCO에 의해 세포 수준에서 본래대로 생물학적 시스템을 해제

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

신경과학

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

안녕하세요 - C : Genomes의 3 차원 구조를 공부하는 방법.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

생물학

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

성인 마우스 난소의 정확한 여포 열거

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

생화학

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...