Este trabalho foi financiado pelo Wellcome Trust e da Fundação Britânica do Coração.

Todos os passos são realizados à temperatura ambiente, salvo indicação em contrário. 1. Enucleação e Fixação de pós-parto Olhos <ol><li> Posicione o mouse sacrificados filhote de cachorro de lado, retire a pele que cobre o olho com uma tesoura. </li><li> Use uma tesoura e uma pinça para enuclear o olho. </li><li> Coloque tesoura abaixo do olho enucleado, cortar o nervo óptico e os tecidos circundantes e retire o olho. Transfira para uma placa de 24 poços contendo 4% de paraformaldeído (PFA) confeccionados em 2x PBS à temperatura ambiente. </li><li> Permitir que cada olho para corrigir por 10 - 15 min, em seguida, transferir para o frio 2x PBS no gelo por 5 - 10 min. O melhor é escalonar enucleações e fixação posterior para garantir o grau de fixação é igual para cada olho. </li></ol> 2. Dissecção da Retinas Murino pós-parto <ol><li> Transferência de olhos em uma placa de Petri contendo 2x PBS usando uma pipeta Pasteur de plástico que foi cortada para alargar o furo e colocar sob um Dissecting microscópio. </li><li> Retire toda a gordura ao redor dos olhos usando uma pinça e tesouras. </li><li> Pierce a borda da córnea com uma tesoura afiada e corte em torno da córnea e íris e descarte. </li><li> Insira fórceps entre retina ea esclera e remover a esclera tomando cuidado para não rasgar a retina. A camada coróide também pode ser removido, mas se houver um risco de danos na retina que ocorra durante este passo, a camada coróide, pode ser deixado em que não deve afectar significativamente a qualidade da imunocoloração. </li><li> Retire a lente eo humor vítreo usando uma pinça. </li><li> Remover os vasos hyaloids reunindo-os em conjunto no centro do olho com uma pinça fina e puxe rapidamente para longe do centro da retina (alternativamente recorte na base de vasos hialóide com uma tesoura fina). </li><li> Enxágüe a retina em forma de copo com PBS em uma placa de Petri limpa. </li><li> Adicione 4-5 incisões radiais que alcançam, aproximadamente, 2/3 do raio da retina utilizando mola Tesourasrs para criar uma forma de &quot;pétala&quot;. </li><li> Desenhe off PBS usando uma pipeta Pasteur para achatar a retina e, em seguida, retire o excesso de PBS com um pequeno pedaço de papel absorvente. </li><li> Lentamente pipeta frio (-20 ° C) queda de metanol por queda sobre superfície de retina até totalmente coberto e, em seguida, inundação com metanol adicional. Isso vai ajudar a consertar as retinas e irá facilitar a permeabilização. As retinas ficará branco. </li><li> Transferir para um tubo de 2 ml usando uma pipeta de Pasteur de plástico que foi cortado para alargar o furo. </li><li> Deixe retina em metanol frio por pelo menos 20 min antes de prosseguir com imunomarcação. Dos anticorpos utilizados aqui, apenas VE-caderina não pode ser utilizado com sucesso em metanol retinas fixos. Neste caso retinas precisa avançar direto para a imunocoloração após a etapa 9. </li><li> Se necessário, as retinas podem ser armazenadas em metanol a -20 ° C durante vários meses antes da coloração. </li></ol> 3. Immuno / coloração isolectin da retina Vasculature <ol><li> Cuidadosamente remover / deitar fora metanol e lavar cuidadosamente as retinas brevemente em PBS (nesta fase a retina ainda está no mesmo tubo de 2 ml a partir da etapa 11). </li><li> Remover PBS e tampa retinas com 100 ul de solução de Perm / Bloco (PBS + 0,3% Triton + BSA a 0,2%) + 5% de soro apropriado (ver Tabela 1) e agitar suavemente durante 1 hora. </li><li> Remover Perm / Bloco retinas e incubar com agitação suave durante a noite a 4 ° C com 100 ul de anticorpos primários seleccionados (ver Tabela 1) e / ou IsolectinB4, todas preparadas por diluição em Perm / bloco. </li><li> Remover anticorpo / isolectin retinas e lavar 4 x 10 min em PBS + 0,3% de Triton (PBSTX). </li><li> Em alguns casos, por exemplo, a seguir a coloração com IsolectinB4-Alexa488 ou a um anticorpo primário directamente conjugados com um anticorpo secundário não é necessária e as retinas podem ser montados directamente (passo 7). Quando um anticorpo primário não conjugado tenha sido utilizado no passo 3, em seguida 100 ul da adequado fluorescent anticorpo secundário (diluição 1/200 em PBSTX) é adicionado e deixado a incubar durante a noite a 4 ° C ou durante 4 horas à temperatura ambiente. </li><li> Remover o anticorpo secundário e lava-se as retinas de 4 x 15 min em 2 ml de PBSTX. </li><li> Transferir retinas em lâminas utilizando uma grande furo pipeta de Pasteur de plástico e remover o excesso de tecido absorvente com PBSTX. Mount retinas pela adição de 50 mL de prolongar montagem sobre uma lamela e posição suavemente sobre a retina. </li><li> Transferir lâminas para frigorífico durante a noite a 4 ° C, assegurando que são planas e protegida da luz, para permitir a montagem prolongar para definir. </li><li> Retina de imagem usando um microscópio confocal ou um microscópio de epifluorescência. </li></ol>

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473 (7347), 298-307 (2011).</li><li>Staton, C. 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=Current+methods+for+assaying+angiogenesis+in+vitro+and+in+vivo.">Current methods for assaying angiogenesis in vitro and in vivo.</a> Int J. Exp. Pathol. 85 (5), 233-248 (2004).</li><li>Goodwin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assays+of+angiogenesis+for+assessment+of+angiogenic+and+anti-angiogenic+agents.">In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents.</a> Microvasc. Res. 74 (2-3), 172-183 (2007).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Dev. Biol. 248 (2), 307-318 (2002).</li><li>Fruttiger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+retinal+vasculature.">Development of the retinal vasculature.</a> Angiogenesis. 10 (2), 77-88 (2007).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat. Protoc. 5 (10), 1659-1665 (1038).</li><li>Mahajan, V. B., Skeie, J. M., Assefnia, A. H., Mahajan, M., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+eye+enucleation+for+remote+high-throughput+phenotyping.">Mouse eye enucleation for remote high-throughput phenotyping.</a> J. Vis. Exp. (57), e3184 (2011).</li><li>Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+the+neonatal+vasculature+and+analysis+of+retinal+angiogenesis+in+mice.">Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.</a> Nat. Protoc. 5 (9), 1518-1534 (2010).</li><li>Allinson, K. R., Lee, H. S., Fruttiger, M., McCarty, J., Arthur, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+expression+of+TGFbeta+type+II+receptor+is+required+to+maintain+vascular+integrity+during+postnatal+development+of+the+central+nervous+system.">Endothelial expression of TGFbeta type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.</a> PLoS One. 7 (9), e39336 (2012).</li><li>Mahmoud, 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=Pathogenesis+of+arteriovenous+malformations+in+the+absence+of+endoglin.">Pathogenesis of arteriovenous malformations in the absence of endoglin.</a> Circ. Res. 106 (8), 1425-1433 (2010).</li><li>Lebrin, F., 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=Thalidomide+stimulates+vessel+maturation+and+reduces+epistaxis+in+individuals+with+hereditary+hemorrhagic+telangiectasia.">Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia.</a> Nat. Med. 16 (4), 420-428 (2010).</li><li>Pi, L., 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=Role+of+connective+tissue+growth+factor+in+the+retinal+vasculature+during+development+and+ischemia.">Role of connective tissue growth factor in the retinal vasculature during development and ischemia.</a> Invest. Ophthalmol. Vis. Sci. 52 (12), 8701-8710 (2011).</li><li>Powner, M. 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=Visualization+of+gene+expression+in+whole+mouse+retina+by+in+situ+hybridization.">Visualization of gene expression in whole mouse retina by in situ hybridization.</a> Nat. Protoc. 7 (6), 1086-1096 (2012).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

O método aqui apresentado oferece uma maneira simples e eficaz para obter imagens das preparações de montagem de retinas inteiras coradas neonatais de rato, tornando-se um modelo facilmente quantificáveis ​​e fisiologicamente relevante da angiogénese. Inicialmente, o olho do rato pode ser bastante difícil para dissecar devido ao seu pequeno tamanho e forma do globo, então um pouco de prática e boas ferramentas de dissecação são necessários para obter resultados confiáveis. Dissecação de olhos preparad...

A angiogénese é um processo de desenvolvimento complexo definida pela formação de novos vasos sanguíneos a partir de uma rede de vasos pré-existentes e é regulada por vários caminhos de sinalização. A mais importante destas é a via de VEGF. VEGF é liberado por células isquêmicos e leva à abertura de angiogênico brotando nos vasos sanguíneos vizinhos. A principal célula endotelial vascular a partir de um novo rebento é uma célula &quot;ponta&quot;, que produz filopodios que chegar em direcção à fonte de VEGF, e é seguida por proliferação de células endoteliais &quot;stalk&quot;. Desta forma, as células endoteliais activadas migrar e proliferar no sentido de uma região avascular, onde formam novos tubos vasculares. A fim de estabilizar esses tubos para apoiar o fluxo sanguíneo, as células endoteliais, pericitos e interagir com as células musculares lisas, que rodeiam os vasos sanguíneos novos 1. A angiogénese é essencial para a vascularização do embrião e os tecidos em crescimento. No entanto, também contribui para muitos patológicasdistúrbios cal, tais como câncer, enquanto os defeitos na angiogênese estão associados com doenças isquêmicas. O desenvolvimento de tratamentos eficazes para regular a angiogénese de drogas como um meio para o tratamento da doença é, portanto, de grande interesse. Por exemplo, os factores anti-angiogénicos eficaz irá reduzir o crescimento do cancro, enquanto pró-angiogénicos estratégias podem ser usadas para tratar a doença isquémica. Assim, os modelos de angiogénese é essencial para compreender melhor a regulação da formação de novos vasos e para o desenvolvimento de novas estratégias terapêuticas para aumentar ou diminuir o crescimento vascular. Um elemento fundamental da pesquisa angiogénese é a escolha de um modelo adequado vascular. Muitos modelos in vitro ter sido concebido para reproduzir os passos básicos da angiogénese, tais como migração de células endoteliais, proliferação e sobrevivência 2,3. Um frequentemente utilizado no ensaio in vitro é a formação de uma rede ramificada de células endoteliais sobre uma matriz Matrigel, apesar de tele não é específico para células endoteliais, nem geralmente incorporam o apoio de pericitos ou de células musculares lisas. Avaliação ex vivo da angiogénese brotando do rato usando a cultura de órgãos, tais como o ensaio de corpo embrióides, o ensaio do anel aórtico e o ensaio metatarso fetal permite a análise da angiogénese de células endoteliais, no contexto de células de suporte adicionais. No entanto, as principais limitações destes ensaios incluem a falta de fluxo sanguíneo e a regressão dos vasos ao longo do tempo, dando uma janela limitada para análise. Portanto, para compreender a regulação da angiogénese, mais precisamente, em modelos in vivo são necessários, tais como os desenvolvidos em ratinhos usando esponjas implantadas subcutaneamente ou tampões matrigel, bem como o modelo de isquemia dos membros posteriores. No entanto, estes modelos são difíceis de analisar devido ao complexo de arquitectura em 3D do neovasos. Em contraste, o embrião do peixe-zebra demonstrou ser um excelente modelo para a visualização de angiogénese no conjunto4 organismo. No entanto, o rato é evolutivamente muito mais intimamente relacionada com a humana do peixe-zebra, tornando o rato um modelo preferido, em muitos casos. Consequentemente angiogénese da retina do rato pós-natal, representa um método bem caracterizado de escolha para a pesquisa da angiogênese. Ele não só proporciona um ambiente fisiológico, mas também um plexo vascular 2D que podem ser facilmente visualizada utilizando manchas fluorescentes adequadas. A arquitetura da rede de vasos, proliferação endotelial, surgimento, o recrutamento de células perivascular e remodelamento podem ser investigadas usando esta técnica. Na retina de rato neonatal, o desenvolvimento dos vasos sanguíneos da retina ocorre na primeira semana de vida pós-natal. Ratos, ao contrário dos humanos, nascem cegos e as retinas só se tornam vascularizado após o nascimento. Vasos retinianos surgir a partir do centro da retina perto do nervo óptico no dia 0 pós-natal (P0), e por angiogénese desenvolver para formar um altamente organizard plexo vascular que atinge a periferia da retina de cerca de 5 P8. Os vasos sanguíneos em desenvolvimento de uma forma característica, com o plexo capilar progressivamente crescente para fora a partir do disco óptico para a periferia para gerar um padrão de alternância regular das artérias e veias, com uma rede capilar interveniente. A vasculatura retiniana fornece um modelo ideal para estudar angiogénese, porque os vasos pode ser facilmente identificado à medida que crescem no que é essencialmente um plano 2D, tornando-se assim relativamente fácil para examinar a vasculatura da retina em preparações plana de montagem 5. Um método para isolar a retina do rato neonatal para análise de respostas de células endoteliais da ponta ao longo de várias horas ex vivo, foi avaliado 6. Alternativamente, uma investigação mais detalhada de todo o sistema vascular da retina e marcadores vasculares associadas requer imunocoloração de amostras retina fixos em diferentes fases de desenvolvimento. Aqui nós fornecemosUma descrição detalhada de um método confiável e tecnicamente simples que usamos para a coloração de montagem todo o plexo vascular retiniana de murino, a partir da enucleação inicial de pós-natal do olho (ver também a 7) por meio dos protocolos de coloração para imagiologia último sob o microscópio.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Plastic pipettes 3 ml</td>
			<td>Scientific Laboratory Supplies</td>
			<td>PIP4210</td>
			<td>Remove tip with scissors to create a wide bore</td>
		</tr>
		<tr>
			<td>BD Falcon 24-well plates</td>
			<td>Scientific Laboratory Supplies</td>
			<td>353226</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Select Petri dishes TV 90 mm</td>
			<td>Scientific Laboratory Supplies</td>
			<td>SLS2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Histobond slides</td>
			<td>VWR</td>
			<td>631-0624</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips 22 x 22 mm</td>
			<td>VWR</td>
			<td>631-1336</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5</td>
			<td>World precision instruments</td>
			<td>14095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spring scissors 10.5 cm long, with straight 8 mm blades</td>
			<td>World precision instruments</td>
			<td>501235</td>
			<td>Used for enucleation</td>
		</tr>
		<tr>
			<td>Vannas scissors 8.5 cm long, with straight 7 mm blades, and 0.025 x 0.015 mm superfine tips</td>
			<td>World precision instruments</td>
			<td>500086</td>
			<td>Used for dissecting retina</td>
		</tr>
		<tr>
			<td>Superfine vannas scissors 8 cm long with straight 3 mm blades, and 0.015 x 0.015 mm superfine tips</td>
			<td>World precision instruments</td>
			<td>501778</td>
			<td>Used for dissecting retina</td>
		</tr>
		<tr>
			<td>crystal clear' tubes 2 ml</td>
			<td>Starlab</td>
			<td>E1420-2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Make up in 2x PBS and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Mounting reagent</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-alexa 594</td>
			<td>Invitrogen</td>
			<td>I21413</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-alexa 488</td>
			<td>Invitrogen</td>
			<td>I21411</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primary Antibodies</td>
		</tr>
		<tr>
			<td>Anti-NG2 (rabbit polyclonal )</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td>Detects pericytes</td>
		</tr>
		<tr>
			<td>Anti-GFAP (clone GA5, mouse monoclonal)</td>
			<td>Millipore</td>
			<td>MAB360</td>
			<td>Detects astrocytes</td>
		</tr>
		<tr>
			<td>Anti-Desmin (clone Y66, rabbit monoclonal)</td>
			<td>Millipore</td>
			<td>04-585</td>
			<td>Detects pericytes</td>
		</tr>
		<tr>
			<td>Anti-Collagen IV (rabbit polyclonal )</td>
			<td>Chemicon</td>
			<td>AB756P</td>
			<td>Detects vascular basement membrane</td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle actin-Cy3 (clone 1A4, mouse monoclonal)</td>
			<td>Sigma</td>
			<td>C6198</td>
			<td>Detects vascular smooth muscle cells.</td>
		</tr>
		<tr>
			<td>Anti-Endoglin (clone MJ7/18, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>550546</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>Anti-CD31 (clone MEC13.3, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>Anti-VE-Cadherin (clone 11D4.1, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>550548</td>
			<td>Detects endothelial cell junctions</td>
		</tr>
		<tr>
			<td>Anti-Alk1 (goat polyclonal)</td>
			<td>R&amp;D Systems</td>
			<td>AF770</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Secondary Antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11012</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-rat-Alexa488</td>
			<td>Invitrogen</td>
			<td>A11006</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-rat-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11007</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-mouse-Alexa488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-goat-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11058</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Microscopes</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Zeiss</td>
			<td>Stemi SV6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Zeiss</td>
			<td>Axiocam HRc</td>
			<td>Camera for dissection microscope</td>
		</tr>
		<tr>
			<td>Epifluorescent Microscope</td>
			<td>Zeiss</td>
			<td>Axioimager with Apotome</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Zeiss</td>
			<td>Axiocam HRm</td>
			<td>Camera for Axioimager</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

whole mount immunofluorescent staining of the neonatal mouse retina to investigate angiogenesis in vivo

A angiogénese é o processo complexo da nova formação de vasos sanguíneos definido pelo surgimento de novos vasos sanguíneos a partir de uma rede de vasos pré-existentes. A angiogénese desempenha um papel importante não só no desenvolvimento normal dos órgãos e tecidos, mas também de muitas doenças em que a formação de vasos sanguíneos é desregulada, tal como o cancro, cegueira e doenças isquémicas. Na vida adulta, os vasos sanguíneos são geralmente de repouso para a angiogênese é um alvo importante para a novela desenvolvimento de drogas para tentar regular a formação de novos vasos especificamente na doença. A fim de entender melhor a angiogênese e desenvolver estratégias adequadas para regulamentá-la, os modelos são necessários que refletem com precisão as diferentes etapas biológicos que estão envolvidos. A retina do rato neonatal fornece um excelente modelo da angiogénese porque as artérias, veias e capilares desenvolver para formar um plexo vascular durante a primeira semana após o nascimento. Este modelo também tem a vantagem de ter um dois-disional estrutura (2D) fazendo análise directa em comparação com o complexo anatomia 3D de outras redes vasculares. Ao analisar o plexo vascular retiniano em diferentes momentos após o nascimento, é possível observar os diferentes estádios da angiogénese sob o microscópio. Este artigo demonstra um procedimento simples para analisar a vascularização da retina de um rato utilizando a coloração fluorescente com anticorpos específicos isolectin e vascular.

Após a dissecção da retina deve ser semelhante a uma flor plana (Figura 1). Usando o protocolo descrito acima, as células endoteliais podem ser vistas por meio da coloração B4-Alexa488 isolectin e apoiando células musculares vasculares são visualizadas através de actina do músculo liso anti-alfa (Figura 2). O ponto de vista de baixa potência usando uma objetiva de 5X na Figura 2 permite uma visão geral da organização vascular da retina. Além disso, usando...

Watch this Scientific Journal Video about Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo at JoVE.com

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

A retina de murino neonatal fornece um modelo fisiológico bem caracterizado da angiogénese, que permite investigações dos papéis dos diferentes genes ou drogas que modulam a angiogénese em<em&gt; In vivo</em&gt; Contexto. Coloração por imunofluorescência de visualizar com precisão o plexo vascular é essencial para o sucesso destes tipos de estudos.

Whole Mount coloração de imunofluorescência do Rato Retina Neonatal para investigar angiogênese<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

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Research

JoVE Journal

Neuroscience

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

45.3K visualizações.  Newcastle University. A retina de murino neonatal fornece um modelo fisiológico bem caracterizado da angiogénese, que permite investigações dos papéis dos diferentes genes ou drogas que modulam a angiogénese em In vivo Contexto. Coloração por imunofluorescência de visualizar com precisão o plexo vascular é essencial para o sucesso destes tipos de estudos.

Vídeo: Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualização do Sistema Nervoso embrionárias no Whole-mount Drosophila Os embriões

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Neurociência

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Dissecação de um olho do rato para um monte inteiro do epitélio pigmentar da retina

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

Em Eletroporação vivo de Retina do rato em desenvolvimento

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Todo-mount imagem de embrião de rato Projeções Sensorial Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Monte todo Dissecção e Imunofluorescência do mouse cóclea Adulto

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch-clamp gravação de células Starburst Amácrinas em um monte de Luxo Preparação de Deafferentated mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Um de uma etapa simples protocolo de dissecação para Whole mount-Preparação do Adulto<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imagem transpupilar de dois fótons in vivo da retina do rato

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Colorindo, Visualização e Análise de Papilas de Fungos, Circunvalolados e Paladar

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...