Este trabalho foi financiado pelo NIH Grant GM59354 atribuído a Sharon E. Bickel. Agradecemos Huy p. Nguyen assistência no desenvolvimento do protocolo para a geração de sondas fluorescentes braço, Ann Lavanway para ajuda com microscopia confocal e J. Emiliano Reed para assistência técnica. Agradecemos também a numerosos colegas na Comunidade da drosófila para discussões úteis e conselhos.

<ol>
	<li>Peters, J. M., Nishiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sister+chromatid+cohesion.">Sister chromatid cohesion.</a> CSH Perspect Biol. 4 (11), (2012).</li><li>McNicoll, F., Stevense, M., Jessberger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cohesin+in+gametogenesis.">Cohesin in gametogenesis.</a> Curr Top Dev Biol. 102, 1-34 (2013).</li><li>Buonomo, S. B., 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=Disjunction+of+homologous+chromosomes+in+meiosis+I+depends+on+proteolytic+cleavage+of+the+meiotic+cohesin+Rec8+by+separin.">Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.</a> Cell. 103 (3), 387-398 (2000).</li><li>Bickel, S. E., Orr-Weaver, T., Balicky, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sister-chromatid+cohesion+protein+ORD+is+required+for+chiasma+maintenance+in+Drosophila+oocytes.">The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes.</a> Curr. Biol. 12 (11), 925-929 (2002).</li><li>Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMC1beta-deficient+female+mice+provide+evidence+that+cohesins+are+a+missing+link+in+age-related+nondisjunction.">SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction.</a> Nat Genet. 37 (12), 1351-1355 (2005).</li><li>Freeman, S. B., 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+National+Down+Syndrome+Project:+design+and+implementation.">The National Down Syndrome Project: design and implementation.</a> Public Health Rep. 122 (1), 62-72 (2007).</li><li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nat Rev Genet. 13 (7), 493-504 (2012).</li><li>Angell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-meiotic-division+nondisjunction+in+human+oocytes.">First-meiotic-division nondisjunction in human oocytes.</a> Am. J. Hum. Genet. 61, 23-32 (1997).</li><li>Tsutsumi, 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=Age-related+decrease+of+meiotic+cohesins+in+human+oocytes.">Age-related decrease of meiotic cohesins in human oocytes.</a> PLoS One. 9 (5), e96710 (2014).</li><li>Liu, L., Keefe, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+cohesin+is+associated+with+age-dependent+misaligned+chromosomes+in+oocytes.">Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes.</a> Reprod Biomed Online. 16 (1), 103-112 (2008).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+predisposes+oocytes+to+meiotic+nondisjunction+when+the+cohesin+subunit+SMC1+is+reduced.">Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced.</a> PLoS Genet. 4 (11), e1000263 (2008).</li><li>Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+weakened+centromere+cohesion+is+a+leading+cause+of+age-related+aneuploidy+in+oocytes.">Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes.</a> Curr. Biol. 20 (17), 1522-1528 (2010).</li><li>Lister, L. 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=Age-related+meiotic+segregation+errors+in+mammalian+oocytes+are+preceded+by+depletion+of+cohesin+and+Sgo2.">Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2.</a> Curr. Biol. 20 (17), 1511-1521 (2010).</li><li>Duncan, F. 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=Chromosome+cohesion+decreases+in+human+eggs+with+advanced+maternal+age.">Chromosome cohesion decreases in human eggs with advanced maternal age.</a> Aging Cell. 11 (6), 1121-1124 (2012).</li><li>Yun, Y., Lane, S. I., Jones, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+dyad+separation+in+meiosis+II+is+the+major+segregation+error+with+maternal+age+in+mouse+oocytes.">Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes.</a> Development. 141 (1), 199-208 (2014).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-mediated+association+of+achiasmate+homologues+declines+with+age+when+cohesion+is+compromised.">Heterochromatin-mediated association of achiasmate homologues declines with age when cohesion is compromised.</a> Genetics. 181, 1207-1218 (2009).</li><li>Jeffreys, C. A., Burrage, P. S., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+system+for+increased+meiotic+nondisjunction+in+older+oocytes.">A model system for increased meiotic nondisjunction in older oocytes.</a> Curr. Biol. 13 (6), 498-503 (2003).</li><li>Weng, K. A., Jeffreys, C. A., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rejuvenation+of+Meiotic+Cohesion+in+Oocytes+during+Prophase+I+Is+Required+for+Chiasma+Maintenance+and+Accurate+Chromosome+Segregation.">Rejuvenation of Meiotic Cohesion in Oocytes during Prophase I Is Required for Chiasma Maintenance and Accurate Chromosome Segregation.</a> PLoS Genetics. 10 (9), e1004607 (2014).</li><li>Perkins, A. T., Das, T. M., Panzera, L. C., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+in+oocytes+during+midprophase+induces+premature+loss+of+cohesion+and+chromosome+segregation+errors.">Oxidative stress in oocytes during midprophase induces premature loss of cohesion and chromosome segregation errors.</a> Proc Natl Acad Sci U S A. 113 (44), E6823-E6830 (2016).</li><li>Dernburg, A. F., Sedat, J. W., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+of+a+role+for+heterochromatin+in+meiotic+chromosome+segregation.">Direct evidence of a role for heterochromatin in meiotic chromosome segregation.</a> Cell. 86, 135-146 (1996).</li><li>Dernburg, A. F., Sedat, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Method Cell Biol. 53, 187-233 (1998).</li><li>Guo, Z., Batiha, O., Bourouh, M., Fifield, E., Swan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Securin,+Separase+and+Cohesins+in+female+meiosis+and+polar+body+formation+in+Drosophila.">Role of Securin, Separase and Cohesins in female meiosis and polar body formation in Drosophila.</a> J Cell Sci. 129 (3), 531-542 (2016).</li><li>Giauque, C. C., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin-Associated+Proteins+HP1a+and+Piwi+Collaborate+to+Maintain+the+Association+of+Achiasmate+Homologs+in+Drosophila+Oocytes.">Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.</a> Genetics. 203 (1), 173-189 (2016).</li><li>Joyce, E. F., Apostolopoulos, N., Beliveau, B. J., Wu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+progenitors+escape+the+widespread+phenomenon+of+homolog+pairing+during+Drosophila+development.">Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development.</a> PLoS Genet. 9 (12), e1004013 (2013).</li><li>Gilliland, W. D., Hughes, S. F., Vietti, D. R., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congression+of+achiasmate+chromosomes+to+the+metaphase+plate+in+Drosophila+melanogaster+oocytes.">Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes.</a> Dev Biol. 325 (1), 122-128 (2009).</li><li>Gyuricza, M. 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=Dynamic+and+Stable+Cohesins+Regulate+Synaptonemal+Complex+Assembly+and+Chromosome+Segregation.">Dynamic and Stable Cohesins Regulate Synaptonemal Complex Assembly and Chromosome Segregation.</a> Curr Biol. 26 (13), 1688-1698 (2016).</li><li>Radford, S. J., McKim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+Imaging+Prometaphase+and+Metaphase+of+Meiosis+I+in+Fixed+Drosophila+Oocytes.">Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes.</a> J Vis Exp. (116), (2016).</li></ol>

Os autores declaram não concorrentes interesses financeiros.

O uso de peixes sondas para avaliar o estado da coesão de braço em prometafase I e metáfase eu Drosophila oócitos é um avanço significativo no campo da drosófila meiose. Historicamente, a drosófila pesquisadores foram limitados ao testes genéticos para inferir a perda prematura da coesão de braço em oócitos maduros11,18,19. Agora, com os métodos apresentados aqui, o estado da coesão do br...

Adequada segregação dos cromossomos durante a mitose e meiose exige que a irmã cromátide coesão ser estabelecido, mantido e lançado em uma coordenada1,2. Coesão é estabelecida durante a fase S e é mediada pelo cohesin complexo, que forma ligações físicas que segure a irmã cromátides irmãs juntos. Na meiose, coesão distal de um crossover também funções para armazenar recombinantes homologs juntos e esta associação física ajuda a garantir a adequada orientação do forma bivalente na metáfase eu do eixo (Figura 1)3,4, 5. Lançamento do braço coesão na anáfase permite-lhe as homologs segregar para polos opostos do fuso. No entanto, se a coesão do braço é perdido prematuramente, recombinantes homologs perderá sua conexão física e segregar aleatoriamente, que pode resultar em gâmetas aneuploid (Figura 1).
Em oócitos humanos, erros na segregação do cromossomo são a principal causa de abortos e defeitos de nascimento, como síndrome de Down,6, e sua incidência aumenta exponencialmente com a idade materna7. Coesão de cromatídeos é criada em oócitos fetais e recombinação meiótica é concluída antes do nascimento. Oócitos depois prenda no meio da prófase eu até ovulação e durante esta prisão, associação física continuada de recombinantes homologs baseia-se na irmã coesão de cromátides irmãs. Portanto, precisa segregação durante a meiose e os resultados de gravidez normal exigem que coesão permanecem intactas por até cinco décadas.
Perda prematura de coesão durante a prolongada detenção meiótica de oócitos humanos tem sido proposta para contribuir para o efeito da idade materna e várias linhas de evidência suportam esta hipótese8,9. No entanto, tendo em conta os desafios da coesão meiótica em oócitos humanos a estudar, muito da nossa compreensão deste fenômeno baseia-se sobre a utilização do modelo organismos5,10,11,12, 13de14,,15.
Drosophila melanogaster oócitos oferecem inúmeras vantagens para o estudo da segregação meiótica de coesão e cromossomo. Um ensaio simples genético permite recuperar a descendência dos gâmetas aneuploid e medir a fidelidade do X-segregação de cromossomos em uma grande escala11,16,17. Além disso, um pode também determinar se erros de segregação do cromossomo surgem porque homologs recombinantes missegregate durante a meiose I, um fenótipo que é consistente com a perda prematura de braço coesão11,18, 19. direta observação do estado de coesão meiótica em oócitos de Drosophila também é possível usar a fluorescência em situ da hibridação (peixe). Apesar de oligonucleotídeos fluorescentes que hibridizam às sequências repetitivas de satélite têm sido usados por mais de uma década para monitorar pericentromeric coesão em madura Drosophila oócitos4,20, análise de braço coesão tem sido muito mais desafiador. Visualização do estado de coesão braço requer uma sonda que se estende por uma grande região de cópia única sequências e é brilhante o suficiente para resultar em sinais visíveis para cada irmã cromátides irmãs quando braço coesão está ausente. Além disso, as condições de fixação do oócito e tamanho dos DNAs rotuladas devem facilitar a penetração21 para o grande oócito maduro de Drosophila (200 µm de largura por 500 µm de comprimento). Recentemente, uma sonda de braço com êxito foi utilizado Visualizar cromátides irmãs oócito de Drosophila durante a anáfase, eu, mas os autores afirmou que não podia detectar um sinal em prometafase ou metáfase prendi oócitos22. Aqui nós fornecemos um protocolo detalhado para a geração de X-braço do cromossoma sondas de peixe e as condições de preparação de oócito que nos permitiram a ensaiar para a perda prematura da irmã coesão cromátide em prometafase I e metáfase eu oócitos. Estas técnicas, que nos permitiram identificar produtos de genes que são necessários para a manutenção da coesão meiótica, permitirá que outros a ensaiar para a irmã coesão cromátide defeitos em oócitos maduros de drosófila de diferentes genótipos.

<table>
	<tbody>
		<tr>
			<td>Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midi Prep kit</td>
			<td>Qiagen</td>
			<td>12143</td>
			<td>Prep BAC clone DNA</td>
		</tr>
		<tr>
			<td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td>
			<td>Sigma</td>
			<td>WGA2</td>
			<td>Amplify BAC clone DNA</td>
		</tr>
		<tr>
			<td>ARES Alexa Fluor 647 DNA labeling kit</td>
			<td>Invitrogen</td>
			<td>A21676</td>
			<td>Label BAC clone DNA</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Remove non-conjugated dye following labeling of BAC clone DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals &#38; Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td>Prepare 10% stock 
			Freeze aliquots</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C75-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Toxic: wear appropriate protection. Prepare 100&#181;g/ml stock in 100% ethanol, store in aliquots at -20 &#176;C. Prepare 1 &#181;g/ml solution in 2X SSCT before use.</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>Sigma</td>
			<td>D-8906</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite</td>
			<td>Drierite Company</td>
			<td>23001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Invitrogen</td>
			<td>15508-013</td>
			<td>Prepare 10 mM stock</td>
		</tr>
		<tr>
			<td>dTTP (10 &#181;mol, 100 &#181;l)</td>
			<td>Boehringer Mannheim</td>
			<td>1277049</td>
			<td>Prepare 1 mM stock</td>
		</tr>
		<tr>
			<td>EDTA (Disodium ethylenediamine tetraacetic acid)</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>Prepare 250 mM stock</td>
		</tr>
		<tr>
			<td>100% ethanol (molecular grade, 200 proof)</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Ultra Pure)</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)</td>
			<td>Sigma</td>
			<td>E-3889</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Ted Pella, Inc.</td>
			<td>18505</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>AM9342</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>901393</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Boehringer Mannheim</td>
			<td>737-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Fisher Scientific</td>
			<td>H350-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore</td>
			<td>HX0603-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydroxylamine</td>
			<td>Sigma</td>
			<td>438227</td>
			<td>Prepare 3 M stock</td>
		</tr>
		<tr>
			<td>4.9 M Magnesium chloride</td>
			<td>Sigma</td>
			<td>104-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 &#376; 7H2O</td>
			<td>Fisher Scientific</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 &#376; 2H2O</td>
			<td>Fisher Scientific</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (0.1mg/ml)</td>
			<td>Sigma</td>
			<td>P8920-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher Scientific</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S209-500</td>
			<td>Prepare 3M stock</td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Polysciences, Inc.</td>
			<td>1131</td>
			<td>Toxic: wear appropriate protection. Prepare 400mM stock</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td>BP327-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>Thermo Scientific</td>
			<td>28320</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>10% Triton X-100</td>
			<td>Thermo Scientific</td>
			<td>28314</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 1 mM EDTA, pH = 8.0</td>
		</tr>
		<tr>
			<td>20X SSC (Saline Sodium Citrate)</td>
			<td></td>
			<td></td>
			<td>3 M NaCl, 300 mM sodium citrate</td>
		</tr>
		<tr>
			<td>2X cacodylate fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA</td>
		</tr>
		<tr>
			<td>1.1X Hybridization buffer</td>
			<td></td>
			<td></td>
			<td>3.3X SSC, 55% formamide, 11% dextran sulfate</td>
		</tr>
		<tr>
			<td>Fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution</td>
		</tr>
		<tr>
			<td>PBSBTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.5% BSA, 0.1% Trition X-100</td>
		</tr>
		<tr>
			<td>PBSTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100</td>
		</tr>
		<tr>
			<td>Extraction buffer (PBSTx + Rnase)</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100, 100 &#181;g/mL RNase</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>2X SSCT + 20% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 40% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 50% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AluI</td>
			<td>New England Biolabs</td>
			<td>R0137S</td>
			<td></td>
		</tr>
		<tr>
			<td>HaeIII</td>
			<td>New England Biolabs</td>
			<td>R0108S</td>
			<td></td>
		</tr>
		<tr>
			<td>MseI</td>
			<td>New England Biolabs</td>
			<td>R0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>MspI</td>
			<td>New England Biolabs</td>
			<td>R0106S</td>
			<td></td>
		</tr>
		<tr>
			<td>RsaI</td>
			<td>New England Biolabs</td>
			<td>R0167S</td>
			<td></td>
		</tr>
		<tr>
			<td>BfuCI</td>
			<td>New England Biolabs</td>
			<td>R0636S</td>
			<td></td>
		</tr>
		<tr>
			<td>100X BSA</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>10X NEB buffer #2</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Restriction enzyme digestion buffer. Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase (TdT) 400 U/&#181;l</td>
			<td>Roche/Sigma</td>
			<td>3333566001</td>
			<td></td>
		</tr>
		<tr>
			<td>TdT buffer</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>Cobalt chloride</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Toxic: wear appropriate protection. Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>RNase A (10 mg/mL)</td>
			<td>Thermo-Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cytology Tools etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>#5 INOX, Biologie</td>
		</tr>
		<tr>
			<td>9&#8221; Disposable glass Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td>Autoclave to sterilize</td>
		</tr>
		<tr>
			<td>Shallow glass dissecting dish</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Deep well dish (3 wells)</td>
			<td>Pyrex</td>
			<td>7223-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium microscope slides</td>
			<td>Fisher Scientific</td>
			<td>22-038-104</td>
			<td>Used to cover deep well dishes</td>
		</tr>
		<tr>
			<td>Frosted glass slides, 25 x 75 mm</td>
			<td>VWR Scientific</td>
			<td>48312-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, 3 x 1 in, 1 mm thick</td>
			<td>Thermo-Scientific</td>
			<td>3051</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 10 x 10 mm, No. 1.5</td>
			<td>Thermo-Scientific</td>
			<td>3405</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needle</td>
			<td></td>
			<td></td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Prolong GOLD mounting media</td>
			<td>Molecular Probes</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed-air in can</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000, spectrophotometer</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at room temperature</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at 4 &#176;C</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven or incubator with rotator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A1RSi laser scanning confoal</td>
			<td>Nikon</td>
			<td></td>
			<td>40X oil Plan Fluor DIC (NA 1.3)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;l microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l PCR tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic container with tight fitting lid</td>
			<td>Various</td>
			<td></td>
			<td>To hold Drierite</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Various</td>
			<td></td>
			<td>disposable wipes</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Various</td>
			<td></td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC purified 5&#39;-labeled oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Cy3-labeled probes that recognize the 359 bp satellite repeat of the X chromosome</td>
		</tr>
		<tr>
			<td>Volocity 3D Image Analysis Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Version 6.3</td>
		</tr>
	</tbody>
</table>

using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes

Em humanos, erros de segregação do cromossomo em oócitos são responsáveis para a maioria dos abortos espontâneos e malformações congénitas. Além disso, como as mulheres envelhecem, o risco de conceber que um feto aneuploid aumenta dramaticamente e este fenómeno é conhecido como o efeito da idade materna. Um requisito para a segregação do cromossomo precisos durante as divisões meióticas é manutenção da irmã coesão de cromátides irmãs durante o período de prófase estendida que a experiência de oócitos. Citológicas em seres humanos e organismos modelo sugerem que a coesão meiótica deteriora-se durante o processo de envelhecimento. Além disso, erros de segregação em oócitos humanos são mais prevalentes durante a meiose I, consistente com a perda prematura da coesão do braço. O uso de organismos modelo é fundamental para desvendar os mecanismos subjacentes a idade-dependente perda de coesão. Drosophila melanogaster oferece diversas vantagens para estudar o Regulamento de coesão meiótica em oócitos. No entanto, até recentemente, apenas testes genéticos estavam disponíveis a ensaiar para a perda de coesão de braço em oócitos de genótipos diferentes ou em diferentes condições experimentais. Aqui, um protocolo detalhado é fornecido usando fluorescência em situ hibridação (peixe) para visualizar diretamente os defeitos na coesão de braço em prometafase I e metáfase prendi Drosophila oócitos. Gerando uma sonda de peixe que se para o braço distal do cromossomo X e coletando pilhas de Z confocal, um pesquisador pode visualizar o número de sinais individuais de peixes em três dimensões e determinar se irmã cromátide braços são separados. O procedimento descrito torna possível quantificar defeitos de coesão braço em centenas de oócitos de Drosophila . Como tal, este método oferece uma importante ferramenta para investigar os mecanismos que contribuem para a manutenção da coesão, bem como os fatores que levam à sua extinção durante o processo de envelhecimento.

A Figura 5 apresenta imagens obtidas com uma sonda de braço que se a região citológica 6E-7B no cromossomo X . Este resulta da sonda em um sinal de que co localiza com o de DAPI, é facilmente distinguível do fundo e tem sido usada com sucesso para quantificar defeitos de coesão braço em diferentes genótipos de19. Quantificação de defeitos de coesão se restringia a prometafase I e metáfase eu estágios; oócitos ant...

Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

Usando FISH de hibridização in situ por fluorescência para monitorar o estado da coesão do braço em oócitos de Drosophila em prometáfase e metáfase I | Desenvolvimento | Biologia | Vídeo

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Uso de hibridização in situ de fluorescência (FISH) para monitorar o estado da coesão do braço em oócitos de Drosophila na prometáfase e na metáfase I

Este manuscrito apresenta um método detalhado para a geração X-cromossomo braço sondas e realizando fluorescência situ hibridação in (peixe) para examinar o estado da irmã coesão cromátide em prometafase e metáfase prendi Drosófila ovócitos. Este protocolo é apropriado para determinar se a coesão de braço meiótica é intacta ou interrompidos em diferentes genótipos.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

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11.3K visualizações.  Dartmouth College. Este manuscrito apresenta um método detalhado para a geração X-cromossomo braço sondas e realizando fluorescência situ hibridação in (peixe) para examinar o estado da irmã coesão cromátide em prometafase e metáfase prendi Drosófila ovócitos. Este protocolo é apropriado para determinar se a coesão de braço meiótica é intacta ou interrompidos em diferentes genótipos.

Vídeo: Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Compreender Organogenesis precoce Usando um Simplificado<em&gt; In Situ</em&gt; Protocolo de hibridação em<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Biologia do Desenvolvimento

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

Multi-alvo Chromogenic Whole-mount<em&gt; In Situ</em&gt; Hibridização para Comparando Expressão Gênica em Domínios<em&gt; Drosophila</em&gt; Os embriões

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

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.

Dissecção e imunofluorescência coloração de cogumelo corpo e fotoreceptor neurônios em cérebros adultos Drosophila melanogaster

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

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Celular agregação ensaios para avaliar a vinculação do entalhe drosófila com Trans-ligantes e sua inibição pelo Cis-ligantes

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

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).

Visualizando o nó e placa Notochordal em Gastrulating embriões de rato usando microscopia eletrônica e imunofluorescência de montagem inteira

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

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

Hibridização In Situ de Embriões Inteiros de Astyanax

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Genotipagem e Quantificação da Coloração de Hibridização in Situ em Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Hibridização em Embriões de Zebrafish e Ensaio de Formação de Tubos em iPSC-CEs para estudar o papel da endoglina no desenvolvimento vascular

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Imagem fluorescente de cálcio e subsequente hibridização em Situ para caracterização do precursor neuronal em xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Um Protocolo para Imunohistoquímica e Distribuição In-situ de RNA dentro do Embrião Drosophila Precoce

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Uso de hibridização in situ de fluorescência (FISH) para monitorar o estado da coesão do braço em oócitos de Drosophila na prometáfase e na metáfase I

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