Name: preparation of rat serum suitable for mammalian whole embryo culture
Uploaded: 2014-08-03T22:00:00
Description: Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

참고 : 동물 실험은 실험 동물의 관리 및 사용을위한 건강 가이드의 국립 연구소에 따라 수행 하였다. 의학의 동북 대학 의과 대학의 동물 실험에 대한위원회는 여기에 기술 된 실험 절차를 승인했다. 1. 마취와 개복술 <ol style="margin-left: 40px;"><li> 혈액 수집의 경우, 특정 병원균이없는 수컷 흰쥐를 사용합니다. 물을 제공하는 동안 적어도 18 시간 동안 쥐를 빨리. 혈액을 ...

<ol>
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WEC 실험 결과의 재현성이 높은 품질의 IC 쥐의 혈청과 정확한 배아 해부 기술 3의 사용에 의존하고 있습니다. 금식은 혈액에있는 포도당의 농도를 표준화 할 필요가 있기 때문에 혈액을 수집하기 전에 금식의 기간을 단축하지 마십시오. 호르몬 수준은 발정주기에 따른 여성 동물에서 변화하기 때문에 암컷 쥐 혈청의 제조에 사용되지 않아야하고, 에스트라 디올과 같은 호르몬 변화는 배아...

모델 동물의 다양한 분자 및 세포 수준에서 발달 메커니즘을 조사하기 위해 발달 생물학에 사용됩니다. 예를 들어, 양서류와 조류 종은 널리 이러한 태아가 어머니의 외부 개발하기 때문에 태아의 직접 조작에 적합한 클래식 모델 동물로 사용되어왔다. 이 동물과는 달리, 포유류의 태아는 엄마의 자궁에서 성장하고, 이후 단계에서의 성장은 자궁의 기능에 결정적으로 의존한다. 따라서, 직접적 초기 단계에서 마우스 및 래트에서 것과 포유류 배아를 조작하는 통상적 어렵다. 1960 년대에, 데니스 새로운 지속적인 산소 공급 및 열 제어 1 WEC 장치를 사용하여 포유류의 전체 배아 문화 (WEC) 기술을 설립했다. WEC에서, 마우스 및 쥐의 배아는 생체, (즉, 자궁의 이상으로) 증가 할 수 있습니다. WEC 기술은 종종 다양한 Chemic의를 추가하여 기형에 사용되었지만AL은 배양액에 화합물이 기술은 또한 포유 동물 2-4에서 고유 발달 메카니즘을 조사하기 위해 다양한 개발 생물학 연구에서 사용되었다. 예를 들어, WEC는 형광 염료 4.0, 세포 이식 (6) 및 리포 펙션 (7) 및 일렉트로 8-13 통한 유전자 도입을 이용하여 야생형과 돌연변이 등의 배아 세포 라벨링 등의 다른 기술과 결합된다. 최근 자궁 조작에 나중 단계에서 쥐의 배아에서 발달 과정을 분석하는 데 사용되었으며, 일렉트로 기술 14 ~ 16과 결합되어 있습니다. 그러나, 이들 기술은 초기 단계 배아에 DNA 용액의 정확한 로컬 주입을 달성 어려움으로 인해 postimplantation 및 midgestation 배아의 조작에 적합하지 않다. 비록 초기 배아 (에 초음파 유도 세포 이식 및 바이러스 벡터를 주입 즉,, E8.5-E-9.5 마우스의) 자궁 내 이전에 17, 18, ​​우수한 기술은 높은 성공률로이 실험을 수행하는 데 필요한보고되었습니다. 따라서, 높은 접근성 생체와 WEC는 마우스와 쥐 배아의 조작에 대한 장점이 있습니다. 수컷 쥐에서 준비 즉시 원심 분리 (IC) 쥐의 혈청은 종종 WEC 매체에 사용됩니다. 배아 (쥐 마우스 나 E10.0에서 E8.0 이전 즉,) postimplantation 단계에서 배양하면, 합성 중간 IC 쥐 혈청의 혼합물은 종종 WEC 19을위한 매체로 사용됩니다. 더 현재 대안 미디어가 할 수 없기 때문에 그러나, 중반 임신에서 문화 배아에 (즉., E8.0 12.5 마우스 배아 또는 쥐의 배아에서 E10.0-E14.5에서), 100 %의 혈청 매체로 사용한다 2 일 이상 체외에서 정상적으로 성장하는 배아. 고품질 쥐 혈청의 제조는WEC 실험에서 재현성을 달성하기위한 중요한 단계. 연기를 원심 분리에 비해, IC는 적혈구의 대부분이 이미 피브린 응괴로부터 분리 되었기 때문에 혈청 피브린 응괴를 짜내 수집 될 때 용혈을 줄이는 이점이있다. 용혈성 쥐의 혈청은 래트 및 마우스 배아의 정상적인 성장을 지원하는 데 실패 할 때, 즉시 원심 분리하여 혈청을 준비 지연 원심 분리를 사용하여 제조하는 것이 바람직하다. 우리의 프로토콜이 다른 프로토콜에 비해 두 단계를 추가로 포함 18 ~ 20 (예., 이전에 첫 번째 원심 분리에 얼음에 수집 된 혈액을 저장하고 처음으로 원심 분리 한 후 4 ° C에서 2 시간 동안 수집 된 혈액 샘플을 유지). 전반 단계는 혈병의 형성을 지연시킬 수 있고, 후자의 단계는 락 스퀴즈 피브린 응괴의 응고를 촉진시킨다. 따라서, 우리의 프로토콜은 초보자를 사용할 수 있습니다. 그러나, 재생 채혈하고 혈청단순히 프로토콜 책을 참조하여 정확하게 추출은 19 ~ 21 매우 어렵습니다. 이 비디오 문서에서는, 우리는 수컷 쥐와 원심 분리하여 혈청을 추출의 복부 대동맥에서 채혈을 포함 최적의 IC 쥐 혈청의 준비를위한 우리의 표준 프로토콜을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

도 1은 혈액 세포 및 혈청의 분리에 개시된 방법의 대표적인 결과를 나타낸다. 우리는 일반적으로 은퇴 한 남성 쥐 (그림 1A)에서 혈액 15 ㎖를 얻을 수 있습니다. 원심 분리에 의해 수집 된 혈액 혈청과 화살표로 표시되는 피브린 응고, 및 혈액 세포 (도 1c)를 포함하는 하부층을 포함하는 상부 층으로 분리 될 수있다. 중요한 사실은, 용혈성 혈액 샘플은 혈청, ?...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

포유류의 전체 배아 문화에 적합 쥐 혈청의 제조

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18 랫 두뇌의 Organotypic 슬라이스 문화

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

연구

생물학

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

성인 쥐 심장 Myocytes의 차 문화

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

터미널 차별 유학을위한 준비 및 랫 렌즈 상피 Explants의 문화

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

전 비켜</em이미징 및 미세 애플 리케이션에 적합> 치킨의 배아 문화 시스템

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

새로운 Multilayered 플라스크를 사용하여 포유류 세포 배양의 스케일 업

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Culturing 인간의 배아와 유도 Pluripotent 줄기 세포에 적합 마우스 배아 Fibroblast 세포의 준비

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

무 혈청 전체 배아 배양 시스템에서 마우스 배아 개발

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

전체 마운트하여 제브라 피쉬 배아 시편 스테인드 관찰 및 분석을위한 평면 마운트 준비<em&gt; 현장에서</em&gt; 하이브리드

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

비 효소, Mammospheres의 자연 발생에 대한 사전 침습 유방 병변의 혈청이없는 조직 문화

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...