Name: zygotic fluorescence recovery after photo-bleaching analysis for chromatin looseness that allows full-term development
Uploaded: 2018-06-12T14:30:00
Description: Watch this Scientific Journal Video about Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development at JoVE.com

Agradecemos Satoshi Kishigami, Sayaka Wakayama, Hiroaki Nagatomo, Satoshi Kamimura e Kana Kishida fornecendo comentários críticos e suporte técnico. Este trabalho foi parcialmente financiado pelo programa do Ministério da educação, cultura, esportes, ciência e tecnologia para promover a reforma das universidades nacionais, m.o.; a sociedade japonesa para a promoção da ciência (16H 02593), Asada Science Foundation e da Fundação de ciência Takeda para TW Os financiadores não tinham qualquer papel no projeto de estudo, coleta de dados e análise, a decisão de publicar ou preparação do manuscrito.

Este estudo foi realizado em estrita conformidade com as recomendações do guia para o cuidado e o uso de animais de laboratório da Universidade de Yamanashi. O protocolo foi aprovado pelo Comité sobre a ética de experimentos Animal da Universidade de Yamanashi (número de licença: A24-50). Todas as cirurgias foram realizadas sob anestesia tribromoetanol, e todos os esforços foram feitos para minimizar o sofrimento. Uma visão geral ilustrada dos procedimentos e horário são mostrados na Figur...

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G., Aoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chromatin+structure+in+mouse+preimplantation+embryos+by+fluorescent+recovery+after+photobleaching.">Analysis of chromatin structure in mouse preimplantation embryos by fluorescent recovery after photobleaching.</a> Epigenetics. 11, 85-94 (2016).</li><li>Meshorer, E., 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=Hyperdynamic+plasticity+of+chromatin+proteins+in+pluripotent+embryonic+stem+cells.">Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells.</a> Dev Cell. 10, 105-116 (2006).</li><li>Boskovic, A., 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=Higher+chromatin+mobility+supports+totipotency+and+precedes+pluripotency+in+vivo.">Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.</a> Genes Dev. 28, 1042-1047 (2014).</li><li>Yamagata, K., Ueda, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+live-cell+imaging+of+mammalian+preimplantation+development+and+derivation+process+of+pluripotent+stem+cells+from+the+embryos.">Long-term live-cell imaging of mammalian preimplantation development and derivation process of pluripotent stem cells from the embryos.</a> Dev Growth Differ. 55, 378-389 (2013).</li><li>Ooga, M., Wakayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+analysis+of+chromatin+looseness+in+mouse+zygotes+that+allows+full-term+development.">FRAP analysis of chromatin looseness in mouse zygotes that allows full-term development.</a> PLoS One. 12, e0178255 (2017).</li><li>Quinn, P., Begley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+human+seminal+plasma+and+mouse+accessory+gland+extracts+on+mouse+fertilization+in+vitro.">Effect of human seminal plasma and mouse accessory gland extracts on mouse fertilization in vitro.</a> Australian journal of biological sciences. 37, 147-152 (1984).</li><li>Chatot, C. 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E., 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=Defective+mitochondrial+translation+differently+affects+the+live+cell+dynamics+of+complex+I+subunits.">Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits.</a> Biochim Biophys Acta. 1807, 1624-1633 (2011).</li><li>Subramanian, V., 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=H2A.Z+acidic+patch+couples+chromatin+dynamics+to+regulation+of+gene+expression+programs+during+ESC+differentiation.">H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.</a> PLoS Genet. 9, e1003725 (2013).</li><li>Hayashi-Takanaka, Y., 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=Tracking+epigenetic+histone+modifications+in+single+cells+using+Fab-based+live+endogenous+modification+labeling.">Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.</a> Nucleic Acids Res. 39, 6475-6488 (2011).</li><li>Yamazaki, T., Yamagata, K., Baba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+and+retrospective+analysis+of+DNA+methylation+in+mouse+preimplantation+embryos+by+live+cell+imaging.">Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging.</a> Dev Biol. 304, 409-419 (2007).</li><li>Puschendorf, M., 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=PRC1+and+Suv39h+specify+parental+asymmetry+at+constitutive+heterochromatin+in+early+mouse+embryos.">PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos.</a> Nat Genet. 40, 411-420 (2008).</li></ol>

Como revelado neste estudo, a análise zFRAP não causa danos críticos para o desenvolvimento do termo, sugerindo que este método é uma ferramenta muito útil para revelar a associação entre eventos moleculares e o potencial do desenvolvimento embrionário. Durante a reprogramação, para as duas câmaras como estado de pilhas do ES, pelo qual o potencial diferencial dessas células torna-se tão elevado como aquele do estágio de duas células, frouxidão de cromatina de mudanças em que comparável com embriões d...

Após a fertilização, a estrutura da cromatina é alterada dinamicamente, e estrutura da cromatina zigóticos é então eventualmente estabelecido1,2. Durante este período, em pró-núcleos paternos, proteínas da cromatina dominante é mudada de protamina na histona. A cromatina resultante é extremamente diferente dos espermatozoides e ovócitos femininos em vários pontos (por exemplo, composição variante de histona, modificação de histona). Assim, a cromatina foi formada é pensada para ser importante para o posterior desenvolvimento embrionário. No entanto, apesar dos esforços para revelar os detalhes da estrutura da cromatina zigóticos durante longos períodos, métodos para avaliar a qualidade de zigotos ou para prever seu desenvolvimento termo na fase de uma célula, analisando sua estrutura da cromatina nunca foram estabelecido.
No estudo anterior, é descoberto que zigotos tem uma estrutura extremamente afrouxada cromatina3. Atualmente, frouxidão de cromatina ou abertura é acreditada para ser um importante factor de diferenciação celular potencial de células estaminais embrionárias (ES)4. Pilhas do ES não apresentam homogeneidade na natureza, mas são bastante heterogêneas; em colônias de células ES, alguns transitoriamente adquirem um maior potencial de diferenciação comparável aos blastômeros de embriões do estágio de duas células. Durante esta transição para a dois-célula como estado, frouxidão de cromatina no ES células no que é comparável com o estágio de duas células de embriões5alterações. Assim, frouxidão de cromatina parece ser importante para o potencial de diferenciação celular e é possível que abrir extensivamente cromatina em zigotos é útil para a avaliação do potencial de desenvolvimento zigóticos.
Imagem ao vivo é uma ferramenta poderosa que permite a análise dos eventos moleculares durante a ontogênese desde que esse método permite o posterior desenvolvimento e até mesmo termo desenvolvimento6. Como um dos métodos de imagem ao vivo, FRAP análise tem sido usado para examinar a frouxidão de cromatina embriões pré-implantação e ES células3,4,5. Se frouxidão zigóticos cromatina pode ser analisado sem um efeito prejudicial sobre o termo desenvolvimento pela análise FRAP, pode ser uma ferramenta valiosa para a avaliação da qualidade dos embriões na fase de uma célula. No entanto, os efeitos sobre o desenvolvimento do termo por esse método experimental não foram examinados. Recentemente, um sistema experimental usando FRAP avaliar frouxidão zigóticos cromatina foi desenvolvido. Porque este era um novo sistema de observação de zigotos, ele foi denominado como zigóticos FRAP (zFRAP). zFRAP não afetou criticamente termo desenvolvimento e tem sido relatado em outro lugar7. Neste relatório, é descrito o protocolo desse método experimental.

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Confocal laser scaning microscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">FV1200</td>
			<td style="width:435px;">FV1000 can be also used for FRAP analysis (reference #7)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Thermo plate</td>
			<td style="width:208px;">Tokai Hit</td>
			<td style="width:119px;">TP-110RH26</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Inverted microscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Micro manupilator</td>
			<td style="width:208px;">Narishige</td>
			<td style="width:119px;">MMO-202ND</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Pieze Micro Micromanipulator</td>
			<td style="width:208px;">Prime tech</td>
			<td style="width:119px;">PMAS-CT150</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">35 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351008</td>
			<td>35 x 100 mm style; for IVF</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">60 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351007</td>
			<td>60 x 15 mm style; for embryo culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">50 mm culture dish</td>
			<td style="width:208px;">Falcom</td>
			<td style="width:119px;">351006</td>
			<td>50 x 9 mm style; for manipulation on the stage</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">50 mm glass bottom dish</td>
			<td style="width:208px;">Matsunami</td>
			<td style="width:119px;">D910400</td>
			<td>50 mm dish, 27 mm &#966; hole size; for FRAP analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Mineral oil</td>
			<td style="width:208px;">Organic spceiality chemicals</td>
			<td style="width:119px;">625071</td>
			<td>For FRAP analysis on the glass bottom dishes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Mineral oil</td>
			<td style="width:208px;">Sigma</td>
			<td style="width:119px;">M8410-1L</td>
			<td>For IVF and embryo culture</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">glass capillary</td>
			<td style="width:208px;">Drummond</td>
			<td style="width:119px;">1-000-1000</td>
			<td>For handling mouse zygotes</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Borosilicate glass</td>
			<td style="width:208px;">Prime tech</td>
			<td style="width:119px;">B100-75-10-PT</td>
			<td>For microinjection of mRNA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">Micropipett puller</td>
			<td style="width:208px;">Sutter instrument</td>
			<td style="width:119px;">P-97/IVF</td>
			<td style="width:435px;">For preparation of the injection or holding neadle (out side diameter: 80 &#181;m, inner diameter: 10 &#181;m)&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Microforge</td>
			<td style="width:208px;">&#160;Narishige</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">mMESSAGE MACHINE SP6 Transcription kit</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM1340</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">Poly (A) tailing kit</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM1350</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">PVP solution 10% (PVP-HTF)</td>
			<td style="width:208px;">IrvineSceientific</td>
			<td style="width:119px;">99311</td>
			<td>HEPES buffered HTF containing 10% PVP</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Sodium HEPES</td>
			<td style="width:208px;">Sigma</td>
			<td style="width:119px;">H3784</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">pTOPO eGFP-H2B</td>
			<td style="width:208px;"></td>
			<td style="width:119px;"></td>
			<td>Template plasmid for eGFP-H2B mRNA (reference #6)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:260px;">NorthernMax Gly Sample loading Dye&#160;</td>
			<td style="width:208px;">Thermo 
			Fisher scientific</td>
			<td style="width:119px;">AM8551</td>
			<td>For electrophoresis of in vitro transcribed mRNA</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Phenol chloroform isamyl alcohol</td>
			<td style="width:208px;">nacalai tesque</td>
			<td style="width:119px;">25970-14</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Chloroform</td>
			<td style="width:208px;">nacalai tesque</td>
			<td style="width:119px;">&#160;08401-65</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Not I</td>
			<td style="width:208px;">TOYOBO</td>
			<td style="width:119px;">NOT-111X</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">Ethachinmate</td>
			<td style="width:208px;">WAKO</td>
			<td style="width:119px;">318-01793</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:260px;">3664 Otical power meter</td>
			<td style="width:208px;">Hioki</td>
			<td style="width:119px;">3664</td>
			<td>&#160;Power meter for laser power</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">Stereomicmicroscope</td>
			<td style="width:208px;">Olympus</td>
			<td style="width:119px;">SZX16</td>
			<td></td>
		</tr>
	</tbody>
</table>

zygotic fluorescence recovery after photo-bleaching analysis for chromatin looseness that allows full-term development

Imagem ao vivo é uma ferramenta poderosa que permite a análise dos eventos moleculares durante a ontogênese. Recentemente, frouxidão de cromatina ou abertura tem demonstrada ser envolvido no potencial de diferenciação celular de células-tronco embrionárias pluripotentes. Anteriormente foi relatado que em comparação com células-tronco embrionárias, zigotos abrigam uma estrutura extremamente afrouxada cromatina, sugerindo sua associação com sua totipotência. No entanto, até agora, isso não foi resolvido se essa estrutura extremamente solta/abrir cromatina é importante para o potencial do desenvolvimento embrionário. No presente estudo, para examinar esta hipótese, foi desenvolvido um sistema experimental no quais zigotos que foram analisados por fluorescência recuperação após o clareamento foto pode desenvolver a termo sem qualquer dano significativo. Importante, este sistema experimental precisa de apenas um aquecedor térmico-placa além de um laser confocal microscópio. As conclusões deste estudo sugerem que recuperação de fluorescência após análise de análise (FRAP) foto-branqueamento pode ser usado para investigar se os eventos moleculares na cromatina zigóticos são importantes para o desenvolvimento do termo.

análise de zFRAP com eGFP-H2B
Devidamente produzida mRNA codificação eGFP-H2B, que é visto como uma banda deslocou causada por poli um rejeito (Figura 2A), foi injetado no citoplasma de zigotos com um corpo polar 2 em 1-3 h após a inseminação (Figura 2B). 8 a 12 h após a inseminação, zigotos com 2 pró-núcleos mostrando a expressão de eGFP-H2B foram coletadas e...

Watch this Scientific Journal Video about Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development at JoVE.com

Zygotic Fluorescence Recovery After Photo-bleaching Analysis for Chromatin Looseness That Allows Full-term Development

Frouxidão de cromatina parece estar envolvido no desenvolvimento potencial dos blastómeros. No entanto, não se sabe se a frouxidão de cromatina pode ser usado como um índice confiável para o potencial do desenvolvimento de embriões. Aqui, tem sido descrito um sistema experimental no qual zigotos de frouxidão-avaliada de cromatina podem desenvolver à gestação completa.

Recuperação de fluorescência zigóticos após o clareamento foto análise por frouxidão de cromatina que permite o desenvolvimento do termo

Live imaging is a powerful tool that allows for the analysis of molecular events during ontogenesis. Recently, chromatin looseness or openness has been ...

The overall goal of this zygotic FRAP or zFRAP is to examine the association between zygotic chromatin looseness and embryonic development potential to term. This method can help answer key questions in the developmental biology field, such as embryonic development. The main advantage of this technique is that it allows the analyzed zygote to develop to full term without significant damage.Visual demonstration of this method is critical as the zFRAP analysis steps are difficult to learn because the experimental condition setting and operation for this analysis are very complex. After preparing eGFP-H2B RNA according to the text protocol, use nuclease-free water to dilute it to 250 nanograms per microliter. In a 60-millimeter dish, place two to three drops each of PVP-HTF, five to six drops of Hepes-buffered CZB, and eGFP-H2B mRNA.Then, use mineral oil to cover the drops. Using a pipette puller, pull borosilicate glass. After breaking the tip, use a microforge to bend the glass pipette to around 30 degrees.Prior to microinjection, confirm that the plate heater on the stage of the micromanipulator is off to prevent killing the zygotes during mRNA injection. Then, using 10%PVP-HTF, wash and coat the inside of the injection needles. Next from the tips fill the glass capillaries with the diluted eGFP-H2B mRNA.Collect the fertilized zygotes with a second polar body, and transfer 20 to 25 of them on to the chamber of the microscope stage. Then, with a holding pipette, hold the zygote and break the zona pellucida and the cytosolic membrane with a piezo drive. Inject about 10 picoliters of mRNA into the cytoplasm of each zygote.Complete the injections within 10 minutes to prevent damage caused by culturing the zygotes outside of the incubator. 10 minutes after microinjection, wash the zygotes in at least three drops of CZB and culture them at 38 degrees Celsius until FRAP analysis. One hour prior to zFRAP analysis, turn on the laser and hot plate attached to the stage of the confocal microscope.Set the temperature to 38 degrees Celsius. On a 60-millimeter glass-bottomed dish, pipette six to 10 microliters of Hepes-buffered CZB medium and cover it with mineral oil. Then, warm the dish on the heater until zygote collection.To make it easy to find the zygote with the confocal microscope, it is important to put the drops throughout the center of the dish. Eight to 12 hours after insemination, collect five to 10 eGFP-H2B expressing zygotes and transfer them to a six to 10-microliter drop of pre-warmed Hepes-buffered CZB medium. To perform a precise quantitative analysis it is important to put the zygote toward the center of the Hepes-buffered CZB drops.Set the bleaching and imaging conditions according to the manufacturer's instructions. For example, under Acquisition Setting, set the scanning speed to four microseconds per pixel and the size to five hundred and twelve. Increase the digital zoom to five, open the DyeList"and choose eGFP"Then, set the observation time to one point six seconds and the number of pictures to 12.Next, start the Stimulus Setting"panel and on UseScanner"click Main"Set the scanning speed to 10 microseconds per pixel. Click Activation in Series"Set PreActivation"to three frames and activation time to five seconds. Then, click Focus x2"and click Stop"Under the stimulus setting panel click Clip Rect"Set the ROI to 40 by 40 pixels.Click Time"and XY"Then, use an optical power meter to access the laser power. It is important to have the lights turned off when measuring the laser. After adjusting the laser gage to set the bleaching and imaging laser power, on the toolbar click Live"and then start Live Plot"Place the oil on the objective lens.Set the chamber on the stage and then find the zygotes in the drops. After finding zygotes, determine the ROI position by clicking Focus x2"And then in the Live Plot"window adjust the HV power gage for the eGFP"channel fluorescence intensity to around 2, 000. To start the zFRAP analysis, click XY"After FRAP analysis, click Series Done"Obtain the FRAP analyzed data, then click on Save As"to save the file 2D View image"as a preferred named file.Use Copy"ROI and Paste"ROI to prepare a reference region and to background respectively. Next, select ROI, REF, and BG, and then in the 2D View"image window, click Series Analysis"Obtain RoFRAP data and then in the Live Plot"window click the Save"button. Carry out data calculations according to the text protocol.Eight to 12 hours after insemination, zygotes with two pronucleai showing the expression of eGFP-H2B"were collected and subjected to zFRAP analysis. After bleaching, the intensity of the eGFP-H2B fluorescent signal recovered slightly because of the inflow of eGFP-H2B into the ROI from the unbleached area in the nucleoplasm. Recovery curves reveal that male pronucleai recovered more quickly and with more mobile fractions than female pronucleai.As demonstrated in these images and graph, after zFRAP analysis, washed and cultured zygotes in CZB medium developed to the blastocyst stage without significant damage. To analyze the full term development, embryos at the two-cell stage were transferred into the ovoducts of pseudo-pregnant mothers. As reported in this table, the birth rate of zFRAP analyzed embryos was slightly, but significantly lower than that of intact embryos but was the same as that of the no zFRAP control.In addition, the pups derived from zFRAP analyzed zygotes seemed healthy and fully developed. While attempting this procedure, it's important to remember to wait for the other expansion. All budget objectorance to avoid focus drift.After watching this video, you should have a good understanding on how to analyze chromatin looseness in zygotes.

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Medir a estabilidade da proteína em Viver embriões Zebrafish Usando Fluorescência Decay Após Fotoconversão (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Biologia do Desenvolvimento

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Snapshot Genome-wide de Reguladores da cromatina e Estados em<em&gt; Xenopus</em&gt; Os embriões por Chip-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Protocolos para obtenção de embriões somáticos e Zygotic para estudar a regulação do desenvolvimento embrionário precoce no Modelo de leguminosas<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

Análise do Cardiomyocyte Desenvolvimento utilizando imunofluorescência em Embryonic Coração do rato

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Análise de Zebrafish larvas Músculo Esquelético Integrity com 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 ...

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Isolamento e Caracterização do mouse Antral Oócitos Baseado em Nucleolar organização da cromatina

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Análise de Desenvolvimento de rim Zebrafish com imagem Time-lapse Usando um microscópio de dissecação Equipado para Seccionamento Optical

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Usando folha de luz microscopia de fluorescência para Imagem Desenvolvimento Zebrafish Eye

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Desenvolvimento de um<em&gt; In Vitro</em&gt; Ensaio para avaliar contrátil Função de células mesenquimais que a transição epitelial-mesenquimal Sofreu

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Análise computacional do Germline Caenorhabditis elegans , para estudar a distribuição de núcleos, as proteínas e o citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...