이 작품은 미국 소아 외과 협회 재단 상 (AG) 및 건강 K08DK098271의 국립 연구소 (AG) bythe 지원되었다.

위스콘신 동물 관리 및 사용위원회의 대학은 모든 절차를 승인했다. 솔루션 1. 준비 <ol><li> 해부 버퍼와 세척 용액으로 1X 인산염 완충 식염수 (PBS)를 사용합니다. </li><li> 병에 설탕과 장소의 30g의 무게로 30 % 자당을 준비합니다. 1X PBS의 99 ML을 추가하고 1 ml의 10 % 아 지드 화 나트륨을 추가합니다. 자당이 모두 용해 될 때까지 잘 혼합. 4 ℃에서 보관이 때까지이 필요합니다. </li><li> 1X PBS, 3 % 소 혈청 알부민 (BSA), 0.1 % 트리톤 X-100을 혼합하여 용액을 차단 준비한다. 철저하게 혼합하고 필요할 때까지 냉장고에 보관합니다. </li><li> 1X PBS로 PFA의 적절한 양을 계량하여 1X PBS에서 8 % 파라 포름 알데히드 (PFA) 용액을 준비하고 완전히 용해 될 때까지 65 ° C에서 배양한다. -20 ° C 냉장고에 25 ㎖ 씩에 보관이 필요할 때까지. 사용 당일 1X PBS로 4 % PFA로 희석. </li></ol> 2. 배아 및 굿 해부이온 <ol><li> 기관 동물 관리 및 사용위원회에 따라 프로토콜을 승인, 초과 임신 한 마우스를 안락사와 PBS 1X 60mm 페트리 접시 포함 얼음 추위에 자궁을 전송합니다. </li><li> 날카로운 가위를 사용하여 해부 현미경, 배아를 노출 열려 자궁 벽을 잘라. PBS 1X 얼음 추위가 포함 된 깨끗한 60mm 페트리 접시에 자궁과 장소에서 배아를 제거합니다. </li><li> 얼음처럼 차가운 1X PBS에서 잘린 각각의 배아를 안락사. 당신이 형광 단백질을 포함하는 형질 전환 마우스를 사용하는 경우, 형광 조명 아래, 긍정적 인 유전자 변형 배아를 식별합니다. </li><li> 해부 현미경을 사용하여 각각의 배아로부터 GI 관 해부. 미세 겸자 사용 왼쪽 위쪽으로 향하게되고, 우측은 배양 접시의 바닥에 대해되도록 배아 배향. 내부 장기를 노출 배아에서 상체 벽을 제거합니다. 등의 신체 벽과 내부 장기 사이의 미세 집게를 삽입합니다. CR배아에서 내부 장기를 제거하기 위해 가위와 같은 절단 작업에서 서로에 대한 집게를 OSS. </li><li> 또한 주변 기관에서 멀리 위장관을 서브 - 해부하고 얼음처럼 차가운 1X PBS를 포함하는 1.5 ml의 microcentrifuge 관에 각각 위장관을 배치합니다. </li></ol> GI 책자 3. 고정 <ol><li> 얼음처럼 차가운 1X PBS로 각각의 위장관 3 회 씻어 후 4 % PFA로 교체. 1.5 시간 동안 실온에서 흔들 플랫폼에 1.5 ml의 마이크로 원심 튜브에서 위장관 책자를 수정합니다. 락 플랫폼에서 1 시간 동안 다음 실온에서 위장관 책자 5 분 동안 3 번 씻어. 이 단계에서 필요할 때까지 1X PBS, 0.1 %의 아 지드 화 나트륨을 함유하는 30 % 수크로오스에서 4 ° C에서 GI 책자를 저장한다. 참고 : 또는 조직의 무결성에 영향없이 1 년에 대해 30 % 자당에 배아를 저장합니다. 30 % 자당의 샘플의 보관 면역 염색 또는 10월에 극저온 sectioni에 대한 하나의 샘플 나중에 처리를 할 수 있습니다NG. 또한, 아래에 설명 된 면역 염색 프로토콜을 진행합니다. </li></ol> 4. 면역 염색 프로토콜 <ol><li> 샘플을 30 % 수크로오스에 저장되어있는 경우에는, 요동 플랫폼상에서 그들 1X PBS에 20 분 동안 3 회 헹군다. </li><li> 실온에서 1 시간에 대한 락을 플랫폼에 솔루션을 차단에 위장 책자를 놓습니다. </li><li> 차단 솔루션을 제거하고 락 플랫폼에서 4 ° C에서 RT 또는 O / N에서 4 시간 중 하나에 대한 차단 솔루션에 희석 일차 항체의 적절한 양의 위장 책자를 품어. (환자에서 얻은 혈청) 인간의 안티 후진타오 항체의 1,000 희석, 1 : 닭 반 녹색 형광 단백질 (GFP) 항체의 1,000 희석 1 : 염소 항 채팅 항체의 100 희석 1을 사용합니다. 주 : 우리는 환자로부터 로컬 얻었다 인간 항 주석은 항체를 이용하지만, 안티 주석은 항체는, 예를 들어 시판되고, 마우스 항 - 주석 (1에서 사용 : 500 희석). </li><li> 1X PBS에서 위장관 책자를 씻어3 시간 5 분 후, 요동 플랫폼상에서 실온에서 1 시간 동안. </li><li> 1 희석 차 항체와 1X PBS를 교체 : (500)를 4 ℃에서 RT 또는 O / N에서 중 4 시간 동안 락을 플랫폼에 솔루션을 차단합니다. 이러한 Dylight, 당나귀 방지 닭 ​​CY2와 당나귀 방지 염소 Cy5에로 당나귀 항 - 인간 아민 반응성 염료를 사용합니다. 당나귀 항 - 마우스의 아민 반응성 염료 405 500 희석 사용하여 마우스 항 - 후진타오 항체는 1을 사용하는 경우. 참고 : 내생 GFP 및 tdTomato 발현과 발달 연령 증가를 면역 염색의 선명도의 강도. 우리는 일반적으로 조직의 효과적인 염색을 위해 또한 사용될 항체의 양을 감소시키기 위해 개별적으로 염색 용액 150 ㎕를 0.2 ㎖의 튜브 용기가 발현 사이 배아. </li><li> 5 분 1X PBS 3 회에서 위장관 책자를 씻어 후 락 플랫폼에서 실온에서 1 시간 동안. </li><li> 유리 슬라이드에 DAPI와 형광 장착 매체의 몇 방울을 배치합니다. 위장관 TRA를 담가형광 장착 매체에 CT 및 조직의 바로 위에 커버 유리를 추가한다. 매체 -G 장착 형광 반영구 시일을 제공 수용성 화합물이다. 참고 : 이러한 Vectashield으로 사용 형광 장착 매체 페이딩과 항체의 광표백 방지 매체를 장착 기반 글리세롤이다. 이러한 제품은 모두 조직을 4 ° C에서 장기간 저장 될 수 있습니다. </li><li> 공 초점 현미경을 사용하여 형광 각각의 이미지를 캡처합니다. 표 3에서 제시 형광 및 사용 필터의 여기 파장을 사용합니다. </li><li> 이미지를 캡처하는 데 사용 촛점의 종류에 따라 적절한 소프트웨어를 사용하여 컴퓨터 보조 이미지 분석을 수행한다. </li></ol>

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The authors have nothing to disclose.

우리의 실험실과 다른 사람은 HSCR의 장 결함도 ganglionated 소장 5,15,16에, aganglionic 콜론으로 제한하지만 근위 확장되지 않는 것으로 나타났습니다. 이러한 변화는 ENS 신경 세포 밀도와 신경 전달 물질 표현형의 변화를 포함 HSCR 환자에서 관찰 된 운동 장애에 대해 설명 할 수있다. 우리는 ENS 형성의 결정을 이해하는 우리의 노력에 위의 기술을 활용했다. 구체적으로는, 이들 기술은 신경 산화...

운동성, 영양소 흡수 및 지방 혈액 흐름을 제어 작동 장용 신경계 (ENS)는, 생명에 필수적 일 것이다. ENS는 마이그레이션 그들이 신경 함유 신경 세포와 신경 교세포로 분화 장을, 식민지 증식 신경 능선 세포 (NCC)에 의해 형성된다. 히 르츠 프룽의 질병 (남자 HSCR, 온라인 멘델의 유전 법칙), 1 4,000에서 출산의 빈도와 multigeneic 선천성 장애, 중단 ENS 형성을 연구의 전형 질병으로 간주 될 수있다. HSCR에서 NCC는 마이그레이션 및 말초 hindgut 2의 가변 길이를 식민지화하는 데 실패. 또한, 다른 일반적인 위장 (GI) 등의 항문 직장 기형, 장 atresias 및 운동 장애와 소아의 발달 결함, 기본 ENS 기능의 장애와 연관되고, 가능성이 미묘한, 과소, 해부학 적 변화와 기능적 변화와 관련이있다ENS 3-6. 따라서, 우리는 ENS 형성의 발달 결정을 이해 할 수 있도록 기술은 병인과 소아 위장관 질환의 치료 가능성을 밝혀 수 있습니다. 마이그레이션 및 식민지에 따라, NCC는 신경 전달 물질의 표현형에 대한 특정 마커 신경 세포로 분화. 콜린성 뉴런 장용성 뉴런 (7)의 대략 60 %를 포함하고, 콜린 아세틸 트랜스퍼 라제 (하는 ChAT), 흥분성 신경 전달 물질 인 아세틸 콜린 (acetylcholine)에 대한 합성 효소에 대한 염색하여 검출 할 수있다. 역사적으로, 콜린성 신경 세포가 중추 신경계에 대한 항체의 항원 특이성을 달리하여 혼동하고 시각화하는 시도 (CNS)는 8 ~ 10 채팅 말초 신경계 (PNS) 대 채팅. 그러나 태반 채팅에 대한 항체 모두 중추 및 말초하는 ChAT 11-13을 인식, 우리는 visua 수 있도록 최근에 설명 된 기술을 가지고고감도 ENS의 콜린성 뉴런의 사용 효율 초기 개발에서 채팅 기자 라인 (14) 달성되었다보다. 여기서는, 해부 뉴런 ENS 신경 전달 물질의 발현을 시각화 뮤린 배아 위장관 고정 및 면역 염색을위한 방법을 제시한다. 생산 tdTomato 동물 채팅-Cre 호텔을, R26R을 : floxSTOP :이 연구를 위해, 우리는 R26R와 성관계 간담 Cre 호텔 쥐를 활용 한 floxSTOP : (원고를 통해 간담 Cre 호텔 tdTomato로 정의) tdTomato 마우스. 이들 동물은 다음 식 (14)하는 ChAT를 검출 형광 리포터를 모두 발현하는 마우스를 얻기 위해, 호모 간담 GFP 리포터 생쥐와 교배시켰다. 이 두 기자 동물 C57BL / 6J 배경에 있습니다 상업적 (잭슨 연구소, 바 하버, ME)을 사용할 수 있습니다.

<table><tbody><tr><td>Phosphate buffered saline</td><td>Oxoid</td><td>BR0014G</td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>S2</td><td></td></tr><tr><td>Sodium azide</td><td>Fisher</td><td>BP9221</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher</td><td>BP1605</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>60 mm Petri dishes</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>SMZ-18 stereoscope</td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>SMZ-18 stereoscope</td><td></td></tr><tr><td>Fine forceps</td><td>Fine science tools</td><td>11252-20</td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>VWR</td><td>20170-038</td><td></td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech, Birmingham, AL</td><td>0100-01</td><td></td></tr><tr><td>Glass slides</td><td>Fisher</td><td>12-550-15</td><td></td></tr><tr><td>Cover glass</td><td>VWR</td><td>16004-330</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Nikon A1</td><td></td></tr><tr><td>Nikon Elements</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

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.

우리는 이전하는 ChAT 식 (14)을 검출하고 tdTomato GFP 형광 리포터를 모두 발현하는 생쥐의 발생을 설명 하였다. floxSTOP : 간단히, 채팅-Cre 호텔 마우스가 R26R와 결합 된 생산 tdTomato 동물 채팅-Cre 호텔을, R26R을 : floxSTOP : (간담 Cre 호텔 tdTomato라고도 함) tdTomato 마우스. 이 동물은 다음 동형 접합 간담 GFP 기자 마우스를 결합했다. 배아를 분리 하였다 및 조직은 고정되기 ...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

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. 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 ...

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11.3K 조회수.  University of Wisconsin School of Medicine and Public Health. 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.

비디오: Immunostaining to Visualize Murine Enteric Nervous System Development

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

마우스의 중추 신경계에서 줄기 세포 주입의 Multimodal 이미징

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

연구

신경과학

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

혈관을 조사하기 위해 신생아 마우스 망막의 전체 마운트 면역 형광 염색<em&gt; 생체 내</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

<em&gt; 체외</em성인 마우스 Myenteric 신경총에서 분리 된 장내 뉴런과 교세포의&gt; 준비

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

쥐과 장내 신경 전구 세포에 대한 양이 세포 이동의 분석

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

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 ...

감염 및 선천성 면역을 연구하기 위한 배아 마우스 뇌 혼합 세포 배양

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

저자 스포트라이트: 감염 및 선천성 면역을 연구하기 위해 배아 마우스 뇌에서 혼합 신경 세포 및 신경교세포 배양 확립  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

감염 및 선천성 면역을 연구하기 위해 배아 쥐 뇌에서 혼합 신경 세포 및 신경교 세포 배양 확립

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

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

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

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

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

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 ...

생물학

Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Source: Quintanilla, L. J., et al. <a href="https://app.jove.com/v/59237">Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

마우스 뇌 절편에서 증식된 신경 줄기세포를 시각화하기 위한 면역형광 염색

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry

	...

쥐 장내 신경 시스템 개발을 시각화하는 면역 염색

요약

더 많은 비디오 탐색

이 비디오의 챕터

마우스의 중추 신경계에서 줄기 세포 주입의 Multimodal 이미징

혈관을 조사하기 위해 신생아 마우스 망막의 전체 마운트 면역 형광 염색

쥐과 장내 신경 전구 세포에 대한 양이 세포 이동의 분석

장용 신경계의 제자리 ^칼슘 이미징에서

감염 및 선천성 면역을 연구하기 위해 배아 쥐 뇌에서 혼합 신경 세포 및 신경교 세포 배양 확립

감염 및 선천성 면역을 연구하기 위해 배아 쥐 뇌에서 혼합 신경 세포 및 신경교 세포 배양 확립

감염 및 선천성 면역을 연구하기 위해 배아 쥐 뇌에서 혼합 신경 세포 및 신경교 세포 배양 확립

담관의 보행을 연구하기위한 Whole-mount Immunohistochemical method의 사용

마우스 뇌 절편에서 증식된 신경 줄기세포를 시각화하기 위한 면역형광 염색