Charline沃克在乔治Streisinger的实验室在俄勒冈大学在20世纪70年代和80年代开发的斑马鱼，在体外受精和雌核发育二倍体的方法。这里所描述的方法是从Charline的协议，该协议是在斑马鱼图书 3不变。 Charline沃克在乔治Streisinger的实验室在俄勒冈大学在20世纪70年代和80年代开发的斑马鱼，在体外受精和雌核发育二倍体的方法。这里所描述的方法是从Charline的协议，该协议是在斑马鱼图书 3不变。

初压概述注：很多，如tricaine，鱼水和生理盐水汉克斯在此协议中所使用的试剂配方是在第10章在网上提供的斑马鱼的<a href="http://zfin.org/zf_info/zfbook/zfbk.html">书</a> 3 （http://zfin.org/zf_info/zfbook/zfbk.html） <ol><li>生产用紫外线灭活精子体外受精的胚胎。 </li><li>充满蛋水的边缘玻璃小瓶（塑料管理单元第）立即将受精卵。盖与乳胶薄板小瓶打开，并捕捉到小瓶在橡胶的顶部（在其中一个洞已被切断）。 </li><li>在液压机的小瓶，在受精后的1.4分钟提高到8000磅（请注意，许多印刷机校准读一个直径为2英寸的活塞上施加的压力，读数必须被转换为PSI）的压力</li><li>受精后6分钟开始，逐渐释放超过1分钟内的压力。 </li></ol>详细的初压过程<ol><li> 实验前一天 <ol style="list-style-type:lower-alpha;"><li>在下午晚些时候，单独的男性和女性受到挤压。可用于任何岁男性，因为他不会是导致遗传物质。女性应该看看大和脂肪在腹部。男性应该看黄色和Spry。 </li><li>保持男性和女性分别持有坦克在那里他们可以很容易地在今天上午访问通宵。 </li></ol></li><li> 实验的早晨<ol style="list-style-type:lower-alpha;"><li>准备Hanks液，添加碳酸氢钠。 </li><li>到鱼设施：冰桶，汉克斯，Tricaine，定时收集精​​子，管。 </li><li>在鱼水稀释Tricaine </li></ol></li><li> 收集精子 <ol style="list-style-type:lower-alpha;"><li>准备一个明确汉克斯的解决方案上的冰小瓶。测量〜50％男性μL受到挤压。 </li><li>一次麻醉tricaine浸泡3或4个男性。他们可以解除麻醉剂用塑料勺。 </li><li>鱼水冲洗和纸巾上（非常重要：水激活精子）的印迹“潮湿的干”。 </li><li>安装与外延，照度低倍率的体视显微镜下一个泡沫的持有人在一个鱼。用血管钳分离的腹鳍和定位在一个microcapillary泄殖腔开幕。 </li><li>中风的双方顺利（Millipore公司）钳轻轻但坚决鱼。 </li><li>由于乳白色的精子来的生殖孔，收集在使用轻柔吸microcapillary。 </li><li>游泳池从冰冷的汉克的生理盐水几个男性的精子。从5-10男性的精子是足够的几百个卵受精。精子在寒冷的汉克斯将继续有效施肥几个小时甚至几天的鸡蛋。 “眼球”的精子浓度，收集到足以使阴天悬挂。 </li></ol></li><li> 紫外线灭活精子 <ol style="list-style-type:lower-alpha;"><li>用冰填充的玻璃培养皿的底部。 </li><li>在盘子里，放在冰的顶部表面玻璃。 </li><li>使用巴斯德吸管，从试管中转移到精子的表面玻璃。要确保它没有采取任何空气气泡的精子进入移液器尽可能。另外，不要泡在精子的表面玻璃，你从吸管中，他们驱逐。驱逐薄，甚至手表玻璃层的精子。丢弃这个移液器。 </li><li>放入培养皿中的紫外线交联剂</li><li>打开2分钟的交联剂</li><li>使用干净的吸管，卸下从表面玻璃的精子，并转移到Eppendorf管标志着紫外线精子置于冰上。 </li></ol></li><li> 蛋的收集和体外授精 <ol style="list-style-type:lower-alpha;"><li>麻醉在三个或四个女性​​在tricaine。 </li><li>鱼水冲洗和印迹在纸巾上的“潮湿的干”。多余的水分会胀大的鸡蛋和防止受精。 </li><li>放置在一个35毫米的塑料培养皿中，用湿布（不湿）手指的女性，按肚皮上轻轻地但坚决。如果她是准备产卵，他们会很容易出来。 </li><li>用抹刀收集鸡蛋和水返回的女性。优良的受精卵是淡黄色，半透明的颜色，而仍然在女性的卵子过长是白色和水样（见下文）。为了确保获得优良的受精卵，他们收集来的鱼设施上的灯后，在第2小时。保持覆盖，以防止干燥鸡蛋。 </li><li>加入30-50μl紫外线精子悬液的鸡蛋，轻轻搅拌枪头。 </li><li>立即LY增加鱼水约1毫升，并启动定时器。左侧的发展在这一点上的鸡蛋会单倍体。 </li></ol></li><li> 。初压请注意：步骤股份公司必须在90秒施肥内完成 ！ <ol style="list-style-type:lower-alpha;"><li>在时刻的计时器开始，鸡蛋加水激活</li><li>稍等片刻，并添加更多的水，漩涡的蛋盘的中心。 </li><li>使用切断和抛光的巴斯德吸管，受精卵转移压力瓶。 </li><li>如果需要，添加更多的鸡蛋水的小瓶，使他们几乎满溢，留在小瓶唇的水珠。 </li><li>放置在水珠的橡胶顶部，并确保在橡胶塑料盖。 </li><li>将小瓶（S）倒挂的压力缸，加入更多的蛋水，以补充气缸。安全放在气缸顶部。 </li><li>将在报刊与柱塞缸和申请8,000磅/平方。英寸</li><li> 6分钟后激活时的总时间4.5分钟的压力，直到记者离开缸。 </li><li>在6分钟后激活，从按删除缸，从缸中删除的小瓶。仔细干燥瓶，以防止冷却的胚胎。 </li><li>复位气缸和填充它与鸡蛋水。 </li><li>准备部分填补他们需要用鸡蛋水未来的小瓶。 </li><li>重复所有批次的鸡蛋。 </li></ol></li></ol>注：气缸将于3瓶一次，所以你可能要延迟几分钟，看看已经在tricaine其他女性鸡蛋的蛋受精。这将有助于加快速度，在同一时间处理多批次。此外，请记住一旦正在使用的记者不要把tricaine在任何更多的鱼，直到治疗完成。让鱼留在麻醉时间过长，可以杀死他们。其次，如果你挤的鱼，而记者正在使用，并获得鸡蛋，他们可能会过于干燥可行的记者，可再次使用时。

<ol>
	<li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+analyses+and+gene-centromere+distances+in+zebrafish.">Segregation analyses and gene-centromere distances in zebrafish.</a> Genetics. 112 (2), 311-311 (1986).</li><li>Streisinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-293 (1981).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Johnson, S. L., Africa, D., Horne, S., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-tetrad+analysis+in+zebrafish:+mapping+the+ros+mutation+and+the+centromere+of+linkage+group+I.">Half-tetrad analysis in zebrafish: mapping the ros mutation and the centromere of linkage group I.</a> Genetics. 139 (4), 1727-1727 (1995).</li><li>Beattie, C. E., Raible, D. W., Henion, P. D., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+pressure+screens.">Early pressure screens.</a> Methods Cell Biol. 237 (71), 2575-25 (1999).</li><li>Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centromere-linkage+analysis+and+consolidation+of+the+zebrafish+genetic+map.">Centromere-linkage analysis and consolidation of the zebrafish genetic map.</a> Genetics. 142 (4), 1277-1277 (1996).</li><li>Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutants+with+disrupted+early+T-cell+and+thymus+development+identified+in+early+pressure+screen.">Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen.</a> Dev Dyn. 237 (9), 2575-2575 (1999).</li></ol>

重要的是要确保用这种方法产生的胚胎，是真正的雌核发育二倍体。作为对照，产生了正常的二倍体卵子（保持预留一些无紫外线灭活精子）和单倍体胚胎（保持不通过EP程序预留的紫外线精子受精卵离合器）离合器的离合器。在1天受精后单倍体胚胎体轴短，不规则的脑和体节的形态。单倍体是不是可行，经过2-3天。 EP二倍体应该看起来像正常的二倍体，虽然一些不规则的“EP怪物”可能会发生?...

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making gynogenetic diploid zebrafish by early pressure

二倍体真核生物的杂合性，往往使繁琐的遗传研究。从杂合子女性产生直接可行的纯合二倍体后代的方法，让有害突变，直接在加速向前遗传屏幕的F1诱变女性进行甄别。 Streisinger等人。<sup&gt; 1,2</sup&gt;描述的方法，使雌核发育二倍体（合子）的斑马鱼，斑马鱼的卵用紫外线灭活精子激活使用物理疗法（热或压力），deploymerize微管和防止第二次减数分裂或第一合子细胞分裂。 “初压（EP）的方法将阻塞在减数分裂II，它在受精后不久发生。 EP方法产生一个可行的胚胎的比例较高，可以发展到肥沃的成年人，雌雄兼用。该方法生成的胚胎，除了那些在减数分裂一纯合突变是在EP离合器之间的着丝粒基因位点的50％和小于1％的端粒基因位点检测，通过重组，从他们的着丝粒分离所有位点纯合子。这种方法是复制逐字从斑马鱼书<sup&gt; 3</sup&gt;。

Watch this Scientific Journal Video about Making Gynogenetic Diploid Zebrafish by Early Pressure at JoVE.com

Making Gynogenetic Diploid Zebrafish by Early Pressure

这是一个产生阻塞后，立即用紫外线灭活精子受精的第二次减数分裂雌核发育二倍体斑马鱼的胚胎（仅来自母亲的遗传贡献的胚胎）的方法。 EP胚胎是不完全纯合的，由于在第一次减数分裂重组，但他们是在所有尚未从他们的着丝粒分离，通过重组位点纯合子。

初压雌核发育二倍体的斑马鱼

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

making-gynogenetic-diploid-zebrafish-by-early-pressure

Research

JoVE Journal

Biology

13.4K 次观看.  University of Oregon. 这是一个产生阻塞后，立即用紫外线灭活精子受精的第二次减数分裂雌核发育二倍体斑马鱼的胚胎（仅来自母亲的遗传贡献的胚胎）的方法。 EP胚胎是不完全纯合的，由于在第一次减数分裂重组，但他们是在所有尚未从他们的着丝粒分离，通过重组位点纯合子。

视频: Making Gynogenetic Diploid Zebrafish by Early Pressure

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

科研

生物学

Brain Banking: Making the Most of your Research Specimens

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1. To achieve this goal 6-10 systematic sections should be probed covering the entire structure. Typically this involves processing 1/5 sections which leaves a significant amount of material unprocessed. In order to maximize the material, we propose an inexpensive method for preserving fixed tissue as part of a long-term storage research plan. As tissue is sliced and processed for the desired stain or antibody, alternate sections should be systematically placed in antigen preserve at -20&deg;C for future processing. Using 24-well plates, sections can be placed in order for future retrieval. Using this method, tissue can be stored and processed for immunohistochemistry over the course of years.

Brain banking and systematic sampling of biological material provides the basis for unbiased stereology and maximizes the potential data obtained from each specimen.

脑银行业：使你的研究样本中，大部分

Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given area of interest1.  To ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

常压LAESI质谱生物组织和生物膜的分子成像

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

在大气压力质谱直接分析单细胞

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...