<meta charset="utf8"></head><body>去除斑马鱼鳞片用于培养和骨骼生物学研究

<ol>
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P., Look, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+the+study+of+human+cancer.">Zebrafish as a model for the study of human cancer.</a> Methods Cell Biol. 105, 309-337 (2011).</li><li>Choe, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+fluorescent+zebrafish+lines+that+have+revolutionized+biomedical+research.">Transgenic fluorescent zebrafish lines that have revolutionized biomedical research.</a> Lab Anim Res. 37, 1-29 (2021).</li><li>Brito, R. S., Canedo, A., Farias, D., Rocha, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+zebrafish+(Danio+rerio)+as+an+emerging+model+system+in+ecotoxicology+and+toxicology:+Historical+review,+recent+advances,+and+trends.">Transgenic zebrafish (Danio rerio) as an emerging model system in ecotoxicology and toxicology: Historical review, recent advances, and trends.</a> Sci Total Environ. 848, 157665 (2022).</li><li>Bergen, D. J. 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J., Fulbright, A. N., Parichy, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/&#946;-catenin+regulates+an+ancient+signaling+network+during+zebrafish+scale+development.">Wnt/&#946;-catenin regulates an ancient signaling network during zebrafish scale development.</a> Elife. 7, e37001 (2018).</li><li>Carnovali, M., Luzi, L., Banfi, G., Mariotti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia+affects+bone+metabolism+in+adult+zebrafish+scale+model.">Chronic hyperglycemia affects bone metabolism in adult zebrafish scale model.</a> Endocr. 54, 808-817 (2016).</li><li>Burns, A. R., Guillemin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scales+of+the+zebrafish:+Host-microbiota+interactions+from+proteins+to+populations.">The scales of the zebrafish: Host-microbiota interactions from proteins to populations.</a> COMICR. 38, 137-141 (2017).</li><li>Vieira, E. F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+freshwater+fish+scale+of+the+species+Leporinus+elongatus+as+adsorbent+for+anionic+dyes:+An+isothermal+calorimetric+study.">The use of freshwater fish scale of the species Leporinus elongatus as adsorbent for anionic dyes: An isothermal calorimetric study.</a> J Therm Anal Calorim. 109 (3), 1407-1412 (2012).</li><li>Ighalo, J. O., Eletta, O. A. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+scale+collagen-A+novel+material+for+corneal+tissue+engineering.">Fish scale collagen-A novel material for corneal tissue engineering.</a> Artif Organs. 36 (9), 829-835 (2012).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+porous+nanocomposite+of+sulfur/carbon+obtained+from+fish+scales+for+lithium-sulfur+batteries.">A novel porous nanocomposite of sulfur/carbon obtained from fish scales for lithium-sulfur batteries.</a> J Mater Chem. 1 (10), 3334-3339 (2013).</li><li>Kumar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+noninvasive+technique+for+rapid+extraction+of+DNA+from+fish+scales.">A noninvasive technique for rapid extraction of DNA from fish scales.</a> IJEB. 45 (11), 992-999 (2007).</li><li>Hutchinson, J. J., Trueman, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+analyses+of+collagen+in+fish+scales:+Limitations+set+by+scale+architecture.">Stable isotope analyses of collagen in fish scales: Limitations set by scale architecture.</a> J Fish Biol. 69 (6), 1874-1880 (2006).</li><li>Kumari, S., Rath, P. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL tubes</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl&#160;</td>
			<td>EMD</td>
			<td>HX0603-4</td>
			<td>For pH stabilization and prepration of PRS</td>
		</tr>
		<tr>
			<td>Concave slide</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vectashield</td>
			<td>H-1200</td>
			<td>For cell nuclei visualization</td>
		</tr>
		<tr>
			<td>Diazonium salt (Fast Blue B)</td>
			<td>Sigma</td>
			<td>D9805</td>
			<td>For AP substrate solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) powder&#160;</td>
			<td>Sigma&#160;</td>
			<td>D5523</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (MS222)&#160;</td>
			<td>Sigma&#160;</td>
			<td>E10521</td>
			<td>For preparation of 0.1% MS222</td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Sigma&#160;</td>
			<td>F4135</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid&#160;</td>
			<td>Fisher&#160;</td>
			<td>A38 212</td>
			<td>For acetate buffer</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>BDH1172-4LP</td>
			<td>For 80% glycerol - storage scales</td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>15630106</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl&#160;</td>
			<td>Sigma</td>
			<td>P217-10</td>
			<td>For preparation of PBS</td>
		</tr>
		<tr>
			<td>Lysotracker</td>
			<td>ThermoFisher</td>
			<td>L7528</td>
			<td>For lysosomes&#160; visualization</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td>For Tris-Maleate buffer&#160;</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma</td>
			<td>319937</td>
			<td>For AP substrate solution</td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide&#160;</td>
			<td>Sigma</td>
			<td>319937</td>
			<td>For Enzyme Substrate Solution</td>
		</tr>
		<tr>
			<td>Na2HPO4&#160;</td>
			<td>EMD</td>
			<td>SX0720-1</td>
			<td>For preparation of PBS</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma&#160;</td>
			<td>S9888</td>
			<td>For preparation of PBS</td>
		</tr>
		<tr>
			<td>NaNO2</td>
			<td>Sigma</td>
			<td>S-2252</td>
			<td>For 4% NaNO2</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td>For preparation of 4% PFA</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Sigma&#160;</td>
			<td>S5881</td>
			<td>For scale fixation and pH stabilization</td>
		</tr>
		<tr>
			<td>Napthol-AS-TR-phosphate</td>
			<td>Sigma</td>
			<td>N6125</td>
			<td>For AP substrate solution</td>
		</tr>
		<tr>
			<td>Napthol-AS-TR-phosphate&#160;</td>
			<td>Sigma</td>
			<td>N6125</td>
			<td>For Enzyme Substrate Solution</td>
		</tr>
		<tr>
			<td>Pararosaniline hydrochloride&#160;</td>
			<td>Sigma</td>
			<td>P3750</td>
			<td>For PRS</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 5000 units penicillin and 5mg streptomyocin/ml</td>
			<td>ThermoFisher&#160;</td>
			<td>15140122</td>
			<td>For scale culture media&#160;</td>
		</tr>
		<tr>
			<td>Personal protective equipment (PPE): disposable nitrile gloves, safety glasses/splash goggles and lab coat.</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td>For scale fixation</td>
		</tr>
		<tr>
			<td>Sodium acetate&#160;</td>
			<td>Sigma&#160;</td>
			<td>S2889</td>
			<td>For acetate buffer</td>
		</tr>
		<tr>
			<td>Standard compound microscope</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir plate</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Tartaric acid</td>
			<td>Sigma</td>
			<td>T6521</td>
			<td>For Tartrate buffer</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Roche</td>
			<td>03 118 142 001</td>
			<td>For Tris-Maleate buffer&#160;</td>
		</tr>
	</tbody>
</table>

in vitro culturing technique for studying cellular dynamics in zebrafish scales

<meta charset="utf8"></head><body>作者聚焦：利用斑马鱼鳞片推进骨细胞活性研究

用于研究斑马鱼鳞片细胞动力学的体外培养技术

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without ...

in-vitro-culturing-technique-for-studying-cellular-dynamics-zebrafish

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

In Vitro Culturing Technique for Studying Cellular Dynamics in Zebrafish Scales

2.5K 次观看.  Mount Saint Vincent University. 我们的研究探讨了骨组织环境干扰的影响。我们对了解模拟微重力和振动对成人和骨骼发育的影响特别感兴趣。我们使用斑马鱼作为首选的模式生物，因为它为生物研究提供了许多优势。与以前的方案相比，我们的方案具有优势，因为它限制了对二氧化碳培养箱的需求，并且它使用标准实验室设备。我们的实验室正试图解开不同类型骨细胞在受...

视频: 作者聚焦：利用斑马鱼鳞片推进骨细胞活性研究 

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

血管血管和血管生成的动力学分析体外

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...