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모든 실험 절차는 도쿄 도립 대학 동물 애호 관리위원회 (IACUC)의 승인을 받았습니다. 실험실 동물 의 관리와 사용을위한 안내서와 동물 보호에 관한 캐나다위원회 의 실험 동물 의 관리와 사용 안내서에 따라 동물을 수용하고 취급했습니다. 우리는 일본 동경 메트로폴리탄 대학교 (Functional Morphology Laboratory) 프런티어 보건 과학부에서 사육되고 유지 된 동물들을 사용했습니다. 1. 동물 배려 및 주거 <ol><li> 폐쇄 형 번식 식민지에서 7 S. murinus (암컷 3 마리와 체중 70 ~ 90g의 4 마리)를 얻습니다. </li><li> 종이 스트립이 들어있는 나무 둥지 상자가있는 플라스틱 케이지에서 이유 후 (출생 후 20 일) 개별적으로 S. murinus를 감금하십시오. 전통적으로 컨디셔닝 된 동물 룸 (23-27 ° C), 습도 조절 장치, 12시 12시 (h : 12시 12 분)의 암흑 사이클을 유지하십시오. 공급 commerc송어 알갱이와 물을 자유롭게 먹는다. </li></ol> 2. 조직 준비 <ol><li> 4 % (w / v) 파라 포름 알데히드 (PFA)를 준비하기 위해 PFA 40g을 0.01M 인산염 완충 생리 식염수 (PBS, pH 7.4) 1,000mL에 녹인다. 흄 찬장에서 용액이 깨끗해질 때까지 와동을 사용하여 혼합하십시오. 4 % PFA O / N를 4 ℃에서 보관하십시오. 주의 : PFA를 취급 할 때는 적절한 개인 보호 장비 (PPE)를 착용하십시오. </li><li> 동물을 관류 및 고정하기 위해 에테르로 마취 한 다음 somnopentyl (pentobarbital sodium, 0.6 mL / kg bodyweight)을 복강 내에 주입하십시오. </li><li> 동물이 완전히 마취 된 후, 메스로 복강을 열고 3cm 정도의 중간 선 복부 절개를 만듭니다. 대동맥을 절개하는 동안 대정맥을 절개하면서이 동맥 분기점 바로 위의 복부 대동맥에 복강 내 카테터를 삽입하여 장골 동맥을 만든다. </li><li> somnopentyl을 주사하십시오 (그래서 pentobarbital1.0 mL / kg 체중). 완전한 안락사 후, 0.01 M PBS (pH 7.4)로 관류시킨 다음 흄 찬장에서 4 % PFA로 관류시킨다. </li><li> 관류 후, 흰 네오프렌 라텍스 (증류수 (DW)가있는 희석 된 흰색 네오프렌 라텍스 3 : 2) 2 ~ 3 mL를 혈관에 라벨을 붙이도록 주사합니다. </li><li> 간, 담낭, 총 담관, 십이지장 및 췌장을 포함한 복부 기관을 관련 신경 및 혈관과 함께 추출합니다. </li><li> 담낭에 약 12 ​​mL의 파란색 네오프렌 라텍스 (희석 한 흰색 네오프렌 라텍스 10 mL에 파란색 잉크 한 방울 넣기)를 담낭에 약 0.1 mL 주입하십시오. </li><li> 전체 마운트 면역 염색을 위해 4 ℃에서 4 % PFA로 밤새 수정하십시오. 주의 : PFA는 유독합니다. PFA를 직접 취급하지 마십시오. PFA는 연기 찬장에서 사용해야합니다. </li></ol> 3. Whole-mount Immunohistochemistry 참고 : 세척, 항체 배양 및 착색을 포함하여 프로토콜 전반에서 the 시편은 4 ℃에서 또는 RT에서 가볍게 흔들어 고정 장치에 남아 있어야합니다 . <ol><li> 1 일 : 잠재적 인 내인성 퍼 옥시 다제의 효소 처리 및 불 활성화 <ol><li> DW 4x로 준비된 시험편을 RT에서 적당한 크기의 유리 병에 1 시간 씩 씻으십시오. PFA를 제거하기 위해 시료를 nutator에서 조심스럽게 흔들어주십시오. 솔루션을 교환 할 때 표본이 손상되지 않도록하십시오. </li><li> 1 % (w / v) 오르토과 요오드 산을 준비하려면 DW 100 mL에 1 g의 오르토 요오드 산을 용해시킨다. </li><li> 내재 peroxidase 반응을 방지하기 위해 RT에서 20 분 동안 1 % orthoperiodic 산성에서 표본을 품어. </li><li> 0.04 mol / L Tris-HCl 완충액 (pH 7.6) 100 mL에 파파인 0.004 g을 녹여 0.004 % (w / v)의 파파인을 준비한다. </li><li> DW로 표본을 RT에서 10 분 동안 씻으십시오. </li><li> 부드러운 흔들림이있는 항온조에서 37 ° C에서 2 시간 동안 새로 준비한 0.004 % 파파인으로 표본을 배양하십시오. </li><li> D로 표본을 씻는다.W에서 50-60 분 동안 처리 하였다. </li><li> 4 ° CO / N에서 4 % PFA에 검체를 보관하십시오. </li></ol></li><li> 2 일째 : 효소 처리 <ol><li> 보관 된 시험편을 DW로 4 시간 동안 RT에서 1 시간 씩 씻으십시오. </li><li> 부드러운 흔들림이있는 항온조에서 37 ° C에서 2 시간 동안 새로 준비한 0.004 % 파파인으로 표본을 배양하십시오. </li><li> DW로 표본을 RT에서 50-60 분 동안 씻으십시오. 4 ° CO / N에서 4 % PFA에 검체를 보관하십시오. </li></ol></li><li> 3 일째 : 동결 치료 <ol><li> 위와 같이 저장된 표본을 DW 4x로 각각 1 시간 동안 RT에서 씻으십시오. </li><li> 2.5 g, 5 g, 10 g의 자당을 각각 0.01 M PBS 100 mL에 녹여 2.5 % (w / v), 5 % (w / v), 10 % (w / v) pH 7.4). </li><li> 2.5 % (w / v), 5 % (w / v) 및 10 % (w / v) 자당에 각각 30 분 동안 표본을 담그십시오. </li><li> -20 ° C에서 30-60 분 동안 표본을 동결시킨 다음 RT에서 완전히 녹입니다. 3x 사이클을 반복하십시오. </li><li> 피2 % Triton X-100 (v / v)을 다시 채우고 0.01 M PBS (pH 7.4) 100 mL에 2 mL의 Triton X-100을 첨가한다. </li><li> 4 % CO / N에서 2 % Triton X-100에 시험편을 보관하십시오. </li></ol></li><li> 주 4 : 일차 항체 배양 <ol><li> BSA (Bovine Serum Albumin) 0.2g, Triton X-100 0.3mL 및 sodium azide 0.1g을 0.01M PBS (pH 7.4) 100mL에 녹여 1 차 항체 희석액을 준비한다. 주의 : 나트륨 아자 이드는 매우 독성이 있습니다. 흡입과 접촉은 피해야합니다. 적절한 PPE를 착용하십시오. </li><li> 1 차 항체 (NFP) 1 : 600을 상기 희석 완충액 (단계 3.4.1)에서 희석한다. 검체가 Nator 항체로 4 ° C에서 3 ~ 6 일 동안 배양하여 표본이 nutator에서 부드럽게 흔들리지 않게하십시오. 더 큰 표본은 배양 시간을 늘려야합니다. </li></ol></li><li> 이차 항체 배양 <ol><li> RT에서 각각 1 시간 동안 PBS 4x에서 검체를 씻어서 항체가 결합되지 않은 항체를 제거합니다. </li&gt; <li> 0.2 g의 BSA와 0.3 mL의 Triton X-100을 0.01 M PBS (pH 7.4) 100 mL에 녹여 2 차 항체 희석액을 준비한다. </li><li> 희석 버퍼 (단계 3.5.2)에서 보조 항체 (peroxidase - 접합, 친화 - 정제 양 항 마우스 IgG - HRP) 1 : 600을 희석. 검체를 2 차 항체와 함께 4 ° C에서 3 일 동안 배양하고 검체를 nutator에 부드럽게 흔들어 놓습니다. </li></ol></li><li> 천연색 <ol><li> 짙은 냄새가 나는 곳에서 0.05 mol / L Tris-HCl 완충액 (pH 7.6) 100 mL에 3,3&#39;- 디아 미노 벤지딘 테트라 히드로 클로라이드 (DAB) 0.002 g을 넣고 용액을 어둡게 유지하여 DAB 착색 용액을 조제한다. </li><li> 각각 1 시간 동안 PBS에서 4x RT로 시료를 씻으십시오. </li><li> 갓 준비된 DAB 착색 용액에 30 % H 2 O 2 10 μL를 넣고 4 ° C / N 용액 또는 3 일 동안 incubate 한 후 nutator에서 부드럽게 흔들어 준다. </li><li> pla로 반응 중지최적의 염색 강도에 도달하면 0.04 % 나트륨 아자 이드로 PBS로 표본을 넣는다. 참고 :이 단계까지 나트륨 아자 이드의 사용을 피하십시오. 이는 양 고추 냉이 퍼 옥시 다제를 억제하기 때문입니다. </li></ol></li><li> 얼룩진 조직 이미징 <ol><li> PBS가 담긴 페트리 유리 접시 (높이 5cm, 직경 10cm)에 조심스럽게 표본을 옮긴다. 실체 현미경에 장착 된 카메라를 사용하여 전체 장착 이미지를 캡처하십시오. 참고 : 전체 마운트 스테인드 조직은 투명 유리 접시에 PBS에 완전히 담근 채로 이미지해야합니다. </li></ol></li></ol>

<ol>
	<li>Kuratani, S., Tanaka, S., Ishikawa, Y., Zukeran, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+hypoglossal+nerve+in+the+chick+embryo+as+observed+by+the+whole-mount+nerve+staining+method.">Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method.</a> Am. J. Anat. 182 (2), 155-168 (1988).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+the+avian+vagus+nerve+with+special+reference+to+the+morphological+innervation+of+heart+and+lung.">Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.</a> Anat. Embryol. (Berl). 182 (5), 435-445 (1990).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+avian+trigeminal+nerves.">Peripheral development of avian trigeminal nerves.</a> Am. J. Anat. 187 (1), 65-80 (1990).</li><li>Yi, S. Q., 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=House+musk+shrew,+Suncus+murinus:+a+novel+and+natural+obesity-resistant+animal+model.">House musk shrew, Suncus murinus: a novel and natural obesity-resistant animal model.</a> Obes. Metab. 6, 22-28 (2010).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+innervation+of+the+gallbladder+in+humans+and+Suncus+murinus+with+special+reference+to+morphological+understanding+of+gallstone+formation+after+gastrectomy.">Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.</a> World J. Gastroenterol. 13 (14), 2066-2071 (2007).</li><li>Yi, S. Q., 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=Anatomical+study+of+the+pancreas+in+the+house+musk+shrew,+Suncus+murinus,+with+special+reference+to+the+blood+supply+and+innervation.">Anatomical study of the pancreas in the house musk shrew, Suncus murinus, with special reference to the blood supply and innervation.</a> Anat. Rec. A Discov. Mol. Cell Evol. Biol. 273 (1), 630-635 (2003).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+the+innervation+of+the+major+duodenal+papilla+in+humans+and+Suncus+murinus,+from+the+perspective+of+preserving+innervation+in+organ-saving+procedures.">Surgical anatomy of the innervation of the major duodenal papilla in humans and Suncus murinus, from the perspective of preserving innervation in organ-saving procedures.</a> Pancreas. 30 (3), 211-217 (2005).</li><li>Yi, S. Q., 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=Surgical+anatomy+of+the+innervation+of+the+pylorus+in+humans+and+Suncus+murinus,+in+relation+to+the+surgical+technique+for+pylorus-preserving+pancreatoduodenectomy.">Surgical anatomy of the innervation of the pylorus in humans and Suncus murinus, in relation to the surgical technique for pylorus-preserving pancreatoduodenectomy.</a> World J. Gastroenterol. 12 (14), 2209-2216 (2006).</li><li>Yi, S. Q., Ren, K., Kinoshita, M., Takano, N., Itoh, M., Ozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+Extrahepatic+Biliary+Tract,+With+Special+Reference+to+the+Direct+Bidirectional+Neural+Connections+of+the+Gall+Bladder,+Sphincter+of+Oddi+and+Duodenum+in+Suncus+murinus,+in+Whole-Mount+Immunohistochemical+Study.">Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.</a> Anat. Histol. Embryol. 45 (3), 184-188 (2016).</li><li>Wang, 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=Neurofilament+proteins+in+axonal+regeneration+and+neurodegenerative+diseases.">Neurofilament proteins in axonal regeneration and neurodegenerative diseases.</a> Neural Regen. Res. 7 (8), 620-626 (2012).</li><li>Wyatt, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+of+the+sphincter+of+Oddi+to+the+stomach,+duodenum+and+gall-bladder.">The relationship of the sphincter of Oddi to the stomach, duodenum and gall-bladder.</a> J. Physiol. 193 (2), 225-243 (1967).</li><li>Mawe, G. M., Kennedy, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duodenal+neurons+provide+nicotinic+fast+synaptic+input+to+sphincter+of+Oddi+neurons+in+guinea+pig.">Duodenal neurons provide nicotinic fast synaptic input to sphincter of Oddi neurons in guinea pig.</a> Am. J. Physiol. 277, G226-G234 (1999).</li><li>Seo, J. H., Cho, S. S., Lee, I. S., Lee, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+neuropeptidergic+properties+of+the+duodenal+neurons+projecting+to+the+gallbladder+in+the+golden+hamster.">Anatomical and neuropeptidergic properties of the duodenal neurons projecting to the gallbladder in the golden hamster.</a> Arch. Histol. Cytol. 65 (4), 317-321 (2002).</li><li>Padbury, R. T., Furness, J. B., Baker, R. A., Toouli, J., Messenger, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+nerve+cells+from+the+duodenum+to+the+sphincter+of+Oddi+and+gallbladder+of+the+Australian+possum.">Projections of nerve cells from the duodenum to the sphincter of Oddi and gallbladder of the Australian possum.</a> Gastroenterology. 104 (1), 130-136 (1993).</li><li>White, J. J., Reeber, S. L., Hawkes, R., Sillitoe, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wholemount+immunohistochemistry+for+revealing+complex+brain+topography.">Wholemount immunohistochemistry for revealing complex brain topography.</a> J. Vis. Exp. (62), e4042 (2012).</li></ol>

저자는 아무런 이해 상충이 없다고 선언합니다.

이 작품은 neurofilament 단백질에 대한 항체를 사용하여 extrahepatic 담즙 관의 innervation의 시각화를위한 실험 절차를 설명합니다. 이 프로토콜은 쿠라 타니 (Kuratani)와 다나카 (Tanaka) 1 , 2 , 3 에 의해 기술 된 프로토콜로부터 수정되었습니다. 이 실험에서는 전체 마운트 염색 접근법이 2 ~ 3 주 동안 계속되므로 ?...

집 사향 갈기 , Suncus murinus , 주문 Insectivora과 가족 Soricidae에 속한다. 이 작은 포유 동물은 동남 아시아 전역에 분포하며 인간의 거주지 또는 소 펜 4 근처에 위치한 잔디와 풀밭에 서식합니다. 이 종은 마우스, 쥐 및 토끼와 같은 다른 실험 동물보다 인간과 더 유사한 일반적인 형태학적인 특성을 나타낸다. 이전에, S. murinus neurofilament protein (NFP)에 대한 말초 신경 마커를 이용한 whole-mount immunostaining은 췌장 6 , 주요 십이지장 유두 7 , 유두 8 , 담낭 5 및 간외 담도 9 에서 말초 신경을 표지하기 위해 사용되었습니다. NFP는 성숙한 신경 세포 뼈대의 일부인 거대 분자 복합체입니다. 신경 필라멘트 관련 단백질NFP와 접합체 사이의 상호 작용을 매개한다. 효소 기능과 링커 단백질의 구조는 NFP 10 에 의해 조절됩니다. NFP 복합체는 NF-L (70 kDa), NF-M (150-160 kDa) 및 NF-H (200 kDa)의 세 가지 폴리 펩타이드로 이루어져 있습니다. NFP는 중추 신경계와 말초 신경계에서 신경 세포의 축색 돌기에서 발견 될 수 있습니다. 항 인간 NFP 항체는 중추 신경계와 말초 신경계의 축삭을 표지하는 것으로 나타났습니다. 해부학 적 및 전기 생리 학적 연구 결과에 의하면 괄약근, 담낭 및 근위부 위장관은 11 , 12 , 13 , 14 로 연결되어 있음이 입증되었습니다. 그러나, 그들의 연결 연결의 형태 학적 특징은 아직 정의되지 않았다. 이 연구는 S. murinus bil의 innervation을 표시하기 위해 whole-mount immunohistochemical 염색법을 사용하는 방법을 보여줍니다NFP 항체가있는 iary tract.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">orthoperiodic acid</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">162-00732</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">papain</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">164-00172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">010-15131</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium azide</td>
			<td style="width:71px;">Nacalai Tesque</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neurofilament protein (NFP) antibody</td>
			<td style="width:71px;">Dako</td>
			<td style="width:119px;">M0762, lot 089, clone: 2F11</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td>
			<td style="width:71px;">MBL</td>
			<td style="width:119px;">Code 330</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">349-00903</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I-0125</td>
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using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

이 작품은 전체 마운트 immunohistochemistry 얼룩 방법을 자세하게 설명합니다, 신경 섬유 단백질 항체를 사용하여 Suncus murinus ( S. murinus 의 담즙 기관의 innervation을 표시하기 위해 &#160; ). 먼저 표본을 S. murinus 에서 해부시키고 4 % 파라 포름 알데히드 (PFA)에 고정시켰다. 둘째, 효소 처리 및 잠재적 인 내재적 퍼 옥시 다제 불 활성화가 수행되었다. 그 다음, 시험편을 1 차 항체, 항 - 신경 필라멘트 단백질 항체에 3-6 일 동안 노출시켰다. 이어서, 양 고추 냉이 과산화 효소가 접합 된 2 차 항체와 함께 항온 배양 하였다. 표적을 3,3&#39;- 디아 미노 벤지딘 (DAB) 기질과 반응시켜 발색 반응을 나타내었다. 이 방법은 모든 내장 기관의 신경 분포 분석에 적용 할 수 있습니다. 또한,이 프로토콜은 다른 뉴런 항체를 테스트하기 위해 적응할 수 있지만, 항체의 최적화es가 먼저 수행되어야합니다. 이 방법은 원래 Kuratani와 Tanaka 1 , 2 , 3 에 의해 도입되었습니다.

그림 1 - 4 는 S. murinus의 간외 담관에서 NFP 양성 신경 섬유에 대한 전형적인 결과를 보여줍니다. NFP에 대한 항체는 간외 담도 ( 그림 1 ), 담낭 ( 그림 2 ), 상부 담관 ( 그림 3 ) 및 십이지장 유두 부위 ( 그림 4 )의 전체 이미지에서 높은 특이도 및 최소 배?...

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Suncus murinus 의 외 간성 담도계에서 neurofilament 단백질 발현을 시각화하기위한 whole-mount immunohistochemical 접근법 . 여기에 제시됩니다. 이 프로토콜은 S. murinus 또는 다른 종의 모든 내장 기관의 innervation을 분석하는 데 사용할 수 있습니다.

담관의 보행을 연구하기위한 Whole-mount Immunohistochemical method의 사용<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

using-whole-mount-immunohistochemical-method-to-study-innervation

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Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K 조회수.  Tokyo Metropolitan University.  Suncus murinus 의 외 간성 담도계에서 neurofilament 단백질 발현을 시각화하기위한 whole-mount immunohistochemical 접근법 . 여기에 제시됩니다. 이 프로토콜은 S. murinus 또는 다른 종의 모든 내장 기관의 innervation을 분석하는 데 사용할 수 있습니다. 

비디오: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Double Whole Mount in situ Hybridization of Early Chick Embryos

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

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

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

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

Method for Whole Mount Antibody Staining in Chick

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

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

칙의 전체 마운트 항체 스테 이닝을위한 방법

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish 전체 마운트 고해상도 더블 형광등 현장에서 하이브 리다이 제이션

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

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, ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

전체 마운트를 사용하여 현장에서</em분자 생물학과 Organismal를 링크> 하이브 리다이 제이션

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

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

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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

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

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

전체 산<em&gt; 현장에서</emE11.5 마우스 태아에 E8.5의&gt; 하이브리드

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

높은 처리량 현장에서</em태아 마우스 로우어 Urogenital 트랙트의 mRNA의 표현 패턴을 특성화> 하이브리드화 방법

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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

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

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

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

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

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