Name: using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus
Uploaded: 2017-06-15T12:30:00.000+00:00
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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>

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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><tbody><tr><td>orthoperiodic acid</td><td>Wako</td><td>162-00732</td><td></td></tr><tr><td>papain</td><td>Wako</td><td>164-00172</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Wako</td><td>010-15131</td><td></td></tr><tr><td>sodium azide</td><td>Nacalai Tesque</td><td>312-33</td><td></td></tr><tr><td>neurofilament protein (NFP) antibody</td><td>Dako</td><td>M0762, lot 089, clone: 2F11</td><td></td></tr><tr><td>peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td><td>MBL</td><td>Code 330</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Wako</td><td>349-00903</td><td></td></tr><tr><td>imidazole</td><td>Sigma</td><td>I-0125</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

쥐 장내 신경 시스템 개발을 시각화하는 면역 염색

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

연구

발생학

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

암 연구에서 치료 및 진단 항체 바이오 유통 평가를 위한 생체내 면역형광 국소화

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

암 연구학

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

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 ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

마우스의 담관 결찰 : 폐쇄성 담즙에 의한 염증성 간 손상과 섬유화의 유도

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

의학

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

장용 신경계의 제자리 칼슘 이미징에서

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

신경과학

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

마우스의 Cajal (ICC) 네트워크의 중간 세포의 시각화

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

생물학

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

뮤린 소장에서 상피 내 림프구의 활력 영상

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

면역학 및 감염병학

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

바다 칠성 장어의 간 변태 동안 담즙의 퇴보를 시각화하는 새로운 방법 명확화 ( Petromyzon 스 marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

뮤린 정밀 절단 간 슬라이스 간 생물학의 Ex Vivo 모델로

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

뮤린 스텔레이트 갠리온의 위치, 해부 및 분석

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

담관의 보행을 연구하기위한 Whole-mount Immunohistochemical method의 사용

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