This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

Todos os experimentos e os procedimentos com animais foram aprovados pelo Comitê Cuidado e Uso da Universidade de Tulane Institucional Animal (IACUC). 1. Configuração Procedimento Cirúrgico <ol><li> instrumentos de autoclave, materiais cirúrgicos e fontes de cultura antes da cirurgia. material cirúrgico para cada rato incluem: 1 cortina, uma cortina com furo pré-cortados (0,5 pol x 1,5 pol) no centro, compressas de gaze, e um resguardo absorvente. Instrumentos cirúrgicos incluem: um bisturi com uma lâmina de número 10, 2 pares de pinças, e um par de tesouras finas. fontes de cultura incluem: uma cortina, um par de pinças, e preparado inserções placa de 6 poços com filtros de policarbonato. </li><li> Esterilizar uma plataforma de Plexiglass, uma fase cirúrgica e um espaço de bancada cirúrgica com 70% de etanol. Mantenha o estágio cirúrgico em um recipiente estéril até o uso. <ol><li> Criar uma fase cirúrgica através da perfuração de um a cerca de 2 em por em um furo no centro de uma placa de cultura de 100 milímetros. Em seguida, use uma lixa para suavizarquaisquer arestas vivas e adicionar uma camada de cola de silicone para as bordas do furo para criar uma superfície elevada para os tecidos. </li><li> Alternativamente, projetar o estágio cirúrgico utilizando software CAD e fazer por 3-D de impressão (Figura 1). </li></ol></li><li> Coloque um resguardo absorvente estéril para baixo e estabelecer uma plataforma de plexiglass em cima dela. Coloque a cortina, sem um orifício pré-cortado, uma almofada aquecida ao longo do lado do resguardo absorvente. </li><li> Pré-aquecido estéril soro fisiológico tamponado com fosfato (PBS), meios de comunicação e solução salina a 37 ° C. media de lugar e PBS em placas de cultura separados no topo da almofada de aquecimento e coloque soro fisiológico em um tubo de 50 mL cônico ao lado da configuração cirúrgico. </li><li> Certifique-se de todos os pacotes são abertos antes do início da cirurgia para garantir a manipulação estéril de todos os materiais. Uma lista completa das ferramentas comuns usados ​​neste procedimento são listados na Tabela de Materiais cirúrgicos específicos e ferramentas. </li></ol> 2. Mesentery colheita Tissue <ol><li> Use ratos Wistar machos adultos (350 ± 25 g; 6 - 8 semanas de idade). Outras tensões e as idades de ratos podem ser substituídos. </li><li> Anestesiar ratos por meio de uma injecção intramuscular de cetamina (80 mg / kg de peso corporal) e xilazina (8 mg / kg de peso corporal). Confirmar o rato está sob anestesia por beliscar entre os dedos para verificar se há uma resposta reflexa; não deve haver nenhum. analgesia preventiva para este procedimento terminal não é necessário. </li><li> Raspar a região abdominal e remover cabelo restante usando creme de depilação. Wipe pele abdominal duas vezes com álcool isopropílico a 70%, seguido de povidona-iodo. Para as toalhitas o cirurgião deve começar no centro do local cirúrgico e mover-se para o exterior da área preparada de uma maneira circular a fim de não se sobrepõem áreas que tenham sido previamente limpo com o mesmo pedaço de gaze estéril ou mecha de algodão estéril. Em seguida, transferir animal para a instalação cirúrgica esterilizada e coloque em cima da plat plexiglassFormato. </li><li> Usando uma lâmina de bisturi, fazer uma 0,75 em - 1,25 na incisão no intestino a partir de 1º de abaixo do esterno. Tenha cuidado para não perfurar o intestino ou mesentério (1 camada de pele, uma camada de tecido conjuntivo, e uma camada de músculo). </li><li> Coloque uma cortina com um buraco pré-cortada sobre a incisão e coloque um estágio cirúrgico esterilizado em cima da cortina. Assegurar as alinha abertura com a incisão. Use aplicadores com ponta de algodão estéreis para localizar e retirar o íleo através da abertura estágio cirúrgico. </li><li> Puxe 6 - 8 janelas mesentérica através do estágio usando aplicadores com ponta de algodão, e tenha cuidado para não tocar as janelas (Figura 1). Os tecidos são tipicamente colhidas a partir da região do ileo do intestino delgado começando perto do ceco. Mantenha tecidos expostos úmido com solução salina estéril aquecida, conforme necessário, utilizando uma seringa estéril para escorrer a solução. </li><li> Eutanásia do rato através de injecção intracardíaca de pentobarbital de sódio (0,2 mL por rato). Antes de retirar-mejanelas senteric, garantir o rato é sacrificado pela palpação do coração; não deve haver nenhum impulso. </li><li> Remover os tecidos mesentério desejados usando uma pinça para agarrar a almofada de gordura e uma tesoura fina para cortar a janela. Deixar uma fronteira de gordura (2 mm), em torno da janela. Lavar tecidos vez em aquecido PBS estéril e uma vez na mídia. </li><li> Voltar íleo exteriorizada para a cavidade abdominal e dispor de animais de acordo com as diretrizes institucionais. </li></ol> 3. Mesentery Cultura de Tecidos de Estudos de lapso de tempo <ol><li> Transferência de fontes autoclavados cultura (ver secção 1.1) e tecidos para uma capela de fluxo laminar estéril. </li><li> Use uma pinça para transferir cada tecido no topo de uma membrana filtro de policarbonato. tecidos garra pelas almofada de gordura para evitar danificar a vasculatura. </li><li> Rapidamente se espalhou o tecido usando a almofada de gordura, tomando cuidado para não tocar na janela. Inverta a inserção com o tecido para dentro da parte inferior de uma placa de 6 poços e cobrir com 3 ml de meio (Figura 1 </strong&gt;). meios típicos utilizados para este procedimento inclui mínima meio essencial (MEM) com 1% de Penicilina Estreptomicina (Penstrep) e 10% de Soro Fetal Bovino (FBS). Os meios podem ser suplementados com outros soros e / ou factores de crescimento para estimular a angiogénese. </li><li> Repita os passos de 3,2-3,3 para cada tecido e cultura em condições de incubadora padrão (5% de CO2, 37 ° C) durante até 5 dias. </li></ol> 4. Imagem Time-Lapse de Mesentery Tissue <ol><li> No dia da imagiologia, suplementar os meios em cada poço com conjugado BSI-LECTINA e incubar em condições padrão de cultura, durante 30 min. Lavar tecidos duas vezes com meio livre de lectina. BSI-LECTINA mancha permanecerá visível no tecido mesentério durante até 3 dias em cultura. </li><li> Transferir a placa para um estágio de microscópio. Identificar os vasos sanguíneos e linfáticos com base na sua morfologia e estrutura de rede. </li><li> Localize uma região de rede desejado em cada tecido e tomar imagens. Tome nota do imaginglocal para assegurar a mesma região será capturado para imagens subsequentes. Se estiver usando um palco motorizado, documentar as coordenadas. </li><li> Retorno tecidos para a incubadora e continuar a cultura até ponto final desejado. Repita os passos 4,1-4,3 conforme necessário dependendo pontos de tempo experimentais desejados. </li></ol> 5. Tissue imunomarcação <ol><li> BSI-lectina Labeling <ol><li> Incubar os tecidos durante 30 min a 37 ° C com 01:40 lectina conjugada com FITC em meios (2,5 ml solução de anticorpo por poço em placas de 6 poços) seguida de duas lavagens com meio. Para lavagens, adicionar mídia e substitua imediatamente. </li></ol></li><li> / Etiquetagem Morto Vivo <ol><li> Incubar os tecidos durante 10 min a 37 ° C com 1: 500 de etídio 2 mM homodímero-1 e 1: 500 calceína AM 1 mM em meio (2,5 ml solução de anticorpo por poço em placas de 6 poços) seguida de duas lavagens com meio. </li></ol></li><li> BSI-lectina / Etiquetagem NG2 <ol><li> tecidos espalhada sobre um microscópio deslizoue (1 - 2 tecidos / lâmina) e deixar secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS (3 x 10 min). </li><li> Para marcação com anticorpos primários incubar tecidos durante 1 h à temperatura ambiente com um anticorpo policlonal de NG2: 100 de coelho e soro de cabra normal a 5% (NGS). Lavar tecidos com PBS (3 x 10 min). </li><li> Para a marcação do anticorpo secundário tecidos incubar durante 1 h à temperatura ambiente com 1: 100 de cabra anti-coelho conjugado anticorpo-CY2 (GAR-CY2) e 5% de NGS. Lavar tecidos com PBS (3 x 10 min). </li><li> Incubar os tecidos durante 30 minutos à temperatura ambiente com lectina 01:40 conjugado com FITC em PBS seguido por duas lavagens com PBS. Para lavagens, adicione PBS e depois substituir imediatamente. </li><li> Para montar os slides, cobrir tecidos com 50:50 PBS e solução de glicerol e lugar lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li><li> LYVE-1 / Etiquetagem PECAM<ol><li> Espalhe tecidos numa lâmina de microscópio (1 - 2 tecidos / lâmina) e deixa-se secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para marcação com anticorpos primários incubar tecidos durante 1 h à temperatura ambiente com 1: anticorpo CD31 monoclonal biotinilado 200 rato e 1: policlonal LYVE-1 anticorpo 100 de coelho em PBS + 0,1% de saponina + albumina de soro bovino a 2% (BSA) + 5% de NGS . Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para a marcação do anticorpo secundário, tecidos incubar durante 1 h à temperatura ambiente com 1: 500 de anticorpo de estreptavidina conjugada com Cy3 e 1: 100 GAR-CY2 em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para montar slides, tecidos de cobertura com 50:50 solução PBS e glicerol e colocar uma lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li><li> BrdU / BSI-lectina Labeling <ol><li> Adicionar 1 mg / mL BrdU aos meios de comunicação e substituir a mídia de tecido com uma solução de BrdU. Incubar durante 2 horas a 37 ° C. </li><li> Espalhe tecidos numa lâmina de microscópio (1 - 2 tecidos / lâmina) e deixa-se secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS (3 x 10 min). </li><li> Desnaturação de DNA de tecidos em HCl 2 M durante 1 h a 37 ° C. Lavar tecidos em PBS + 0,1% de saponina (3 x 10 min). </li><li> Para marcação com anticorpos primários, tecidos incubar durante 1 h à temperatura ambiente com 1: 100 monoclonal de ratinho anti-BrdU em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para a marcação do anticorpo secundário, tecidos incubar durante 1 h à temperatura ambiente com 1: 100 de anticorpo de cabra anti-rato Cy3-conjugado (GAM-Cy3) em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li&gt; tecidos Incubar durante 30 minutos à temperatura ambiente com lectina 01:40 conjugado com FITC em PBS seguido por duas lavagens com PBS. </li><li> Para montar slides, tecidos de cobertura com 50:50 PBS e solução de glicerol e lugar lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li><li> BSI-lectina / rotulagem CD11b <ol><li> Espalhe tecidos numa lâmina de microscópio (1 - 2 tecidos / lâmina) e deixa-se secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para marcação com anticorpos primários tecidos incubar durante 1 h à temperatura ambiente com 1: 100 de murganho anti-rato de CD11b em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para a marcação do anticorpo secundário tecidos incubar durante 1 h à temperatura ambiente com 1: 100 GAM-Cy3 em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS+ 0,1% de saponina (3 x 10 min). </li><li> Incubar os tecidos durante 30 minutos à temperatura ambiente com lectina 01:40 conjugado com FITC em PBS seguido por duas lavagens com PBS. </li><li> Para montar slides, tecidos de cobertura com 50:50 PBS e solução de glicerol e lugar lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Este protocolo documenta um método para usar o modelo de cultura de mesentério de rato como uma ferramenta ex vivo para geração de imagens de lapso de tempo de crescimento da rede microvascular. Trabalhos anteriores em nosso laboratório estabeleceu a utilização do nosso modelo para 1) angiogênese 8, 2) lymphangiogenesis 8, 3) interações celulares pericyte-endotelial 8 e 4) teste de drogas anti-angiogênico 9.</su...

O crescimento da rede microvascular e remodelação são denominadores comuns para a função do tecido, cicatrização de feridas, e patologias múltiplas e um processo chave é a angiogénese, definido como o crescimento de novos vasos sanguíneos a partir dos existentes 1, 2. Para Engenharia de Tecidos novos navios ou projetar terapias baseadas angiogénicos, compreendendo a importância da dinâmica celulares envolvidos na angiogênese é crítica. No entanto, este processo é complexo. Ela pode variar em locais específicos dentro de uma rede microvascular e envolve vários tipos de células (isto é, células endoteliais, células musculares lisas, pericitos, macrófagos, as células estaminais) e múltiplos sistemas (redes linfáticas e redes neurais). Embora os modelos in vitro têm contribuído enormemente para examinar a relação entre diferentes células envolvidas na angiogênese 3, a sua relevância fisiológica pode ser prejudicada devido à thei<html> r complexidade limitada eo fato de que eles não refletem de perto um cenário in vivo. Para superar estas limitações, os sistemas de cultura tridimensionais 3, ex vivo modelos de tecido 4, sistemas de microfluidos 5, 6, 7 e modelos computacionais foram desenvolvidos e introduzidos nos últimos anos. No entanto, ainda existe uma necessidade para um modelo com capacidade de lapso de tempo para investigar a angiogénese em redes microvasculares intactas ex vivo. A criação de novos modelos de lapso de tempo para estudos de angiogênese com esse nível de complexidade irá fornecer uma ferramenta inestimável para compreender os mecanismos subjacentes que regulam a angiogênese e para melhorar terapias. Um modelo de potencial que permite a investigação ex vivo da angiogênese através de uma rede microvascular intacta é o modelo de cultura de mesentério de rato&gt; 8. Em trabalho recente, temos demonstrado que o sangue e redes microvasculares linfáticos permanecer viável após o cultivo. Mais importante, o modelo de cultura de mesentério de rato pode ser usado para investigar as interações funcionais pericyte-endotelial de células, sangue e conexões das células endoteliais linfáticas e de imagem time-lapse. O objetivo deste trabalho é fornecer nosso protocolo para o método de imagem time-lapse. Os resultados representativos documentar os vários tipos de células que permanecem viáveis ​​após a estimulação de angiogénese com soro e oferecem exemplos da utilização deste método para a quantificação de respostas angiogénicas específicas dos tecidos, bem como estudos de acompanhamento de células endoteliais.

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			<td height="34" style="height:34px;width:165px;">Drape</td>
			<td style="width:183px;">Cardinal Health</td>
			<td style="width:132px;">4012</td>
			<td style="width:199px;">12&#8221;x12&#8221; Bio-Shield Regular Sterilization Wraps</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Scalpel Handle</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-9843</td>
			<td style="width:199px;">Scalpel Handle, #3; Solid; 4&#34; Length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Surgical Blade</td>
			<td>Cincinnati Surgical</td>
			<td>0110</td>
			<td>Stainless Steel; Size 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Dish (60mm)</td>
			<td>Thermo Scientific</td>
			<td>130181</td>
			<td>10/Sleeve</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:165px;">Graefe Forcep (curved tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5135</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Graefe Forcep (straight tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5130</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:165px;">Noyes Micro Scissor</td>
			<td style="width:183px;">Roboz Surgical Instrument</td>
			<td style="width:132px;">RS-5677</td>
			<td style="width:199px;">Noyes Micro Dissecting Spring Scissors; 
			Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2&#34; Overall Length</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Gauze Pads</td>
			<td>FisherBrand</td>
			<td>13-761-52</td>
			<td style="width:199px;">Non-Sterile Cotton Gauze Sponges; 4&#34;x4&#34; 12-Ply</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Cotton-Tippled Applicators</td>
			<td>FisherBrand</td>
			<td>23-400-124</td>
			<td style="width:199px;">6&#34; Length; Wooden Shaft; Single Use Only</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">6-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-49</td>
			<td style="width:199px;">Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Syring 5ml</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td>Luer-Lok Tip</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Sterile Bowl</td>
			<td>Medical Action Industries Inc.</td>
			<td>01232</td>
			<td style="width:199px;">32 oz. Peel Pouch; Blue; Sterile Single Use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:165px;">6-Well Plate Inserts (CellCrown Inserts)</td>
			<td>SIGMA</td>
			<td>Z681792-3EA</td>
			<td>6-Well Plate Inserts; Non-Sterile</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Polycarbonate Filter Membrane</td>
			<td>SIGMA</td>
			<td>TMTP04700</td>
			<td style="width:199px;">Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&#160;&#181;m, 47&#160;mm, White Plain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Beuthanasia</td>
			<td style="width:183px;">Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 011168</td>
			<td style="width:199px;">Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium&#160;</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Ketamine</td>
			<td style="width:183px;">Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Kateset 100 mg/ml</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Xylazine</td>
			<td style="width:183px;">LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Anased 100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td>2F7122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Invitrogen</td>
			<td>11095080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Saponin&#160;</td>
			<td>SIGMA</td>
			<td>S7900-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>S25372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Povidone-Iodine</td>
			<td>Operand</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hydrochloric Acid</td>
			<td>SIGMA</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Methanol</td>
			<td>Fisher Scientific</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">FITC-conjugated Lectin</td>
			<td>SIGMA</td>
			<td>L9381-2MG</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td>
			<td>SIGMA</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PECAM (CD31) Antibody</td>
			<td>BD Biosciences</td>
			<td>555026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LYVE-1 Antibody</td>
			<td>AngioBio Co.</td>
			<td>11-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit Cy2-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-585-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-227-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-2&#39;-Deoxyuridine</td>
			<td>SIGMA</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Monoclonal Mouse Anti-Bromodeoxyuridine&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Clone Bu20a</td>
			<td>Dako</td>
			<td style="width:132px;">M074401-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Rat CD11b&#160;</td>
			<td>AbD Serotec</td>
			<td>MCA275R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

A angiogénese, definido como o crescimento de novos vasos sanguíneos a partir de vasos pré-existentes, envolve células endoteliais, pericitos, células musculares lisas, células do sistema imunológico, bem como a coordenação com vasos linfáticos e nervos. A multi-celular, as interacções multi-sistema exige a investigação de angiogénese num ambiente fisiologicamente relevante. Assim, enquanto o uso de modelos de cultura celular in vitro proporcionaram conhecimentos mecanicistas, uma crítica comum é que eles não recapitular a complexidade associada com uma rede microvascular. O objetivo deste protocolo é o de demonstrar a capacidade de fazer comparações de lapso de tempo de redes microvasculares intactas antes e após a estimulação da angiogénese em tecidos mesentério de rato cultivadas. tecidos cultivados contêm redes microvasculares que mantêm a sua hierarquia. imunohistoquímica confirma a presença de células endoteliais, células musculares lisas, pericitos, vasos sanguíneos e vasos linfáticos. Em umddition, rotulando tecidos com BSI-lectina permite a comparação de lapso de tempo de regiões de rede local antes e após a estimulação fator de soro ou crescimento caracterizada pelo aumento da germinação capilar e densidade de vasos. Em comparação com os modelos de cultura de células comuns, este método proporciona uma ferramenta para estudos de linhagens de células endoteliais e avaliação de medicamentos angiogénica do tecido específico em redes microvasculares fisiologicamente relevantes.

Após 3 dias em cultura, os tecidos foram marcados com um kit de viabilidade / citotoxicidade Live / morto para demonstrar a viabilidade da microcirculação no mesentério de ratos modelo de cultura (Figura 2A). A maioria das células presentes no mesentério permaneceram viáveis ​​na cultura em que as células endoteliais foram identificados com base na sua localização em segmentos microvasculares. Proliferação de células endoteliais foi também confirmada po...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

A<em&gt; Ex Vivo</em&gt; Método for Imaging Time-Lapse of cultivadas Rato Mesentéricas microvasculares Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

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Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K visualizações.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

Vídeo: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Captura de Reparo de Tecidos em Zebrafish larvas com a Time-lapse Brightfield lupa estereoscópica

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Biologia do Desenvolvimento

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

analisando<em&gt; In Vivo</em&gt; Migração celular usando transplantes de células e de imagem Time-lapse em embriões Zebrafish

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

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

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

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

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

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Um Sanduíche papel de filtro submerso por longo prazo<em&gt; Ex Ovo</em&gt; Imagem Time-lapse de galinha precoce embriões

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Fóton multi tempo lapso imagem para visualizar o desenvolvimento em tempo real: visualização de migrar as células da crista Neural em embriões de peixe-zebra

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Cultivo de células epiteliais de pigmento Retinal em um modelo Ex Vivo da membrana de Bruch humano envelhecido

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Um método In Vivo para estudar a integridade de barreira de sangue-testículo de rato

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Análise da dinâmica da Actomiosina em escalas celulares e teciduais locais usando filmes de lapso de tempo das câmaras de ovos de Drosophila cultivadas

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

Perfusão ex vivo da placenta de roedores

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Um método de montagem em camadas para a microscopia confocal de lapso de tempo prolongado de embriões zebrafish inteiros

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...