Este trabalho foi apoiado pela Deutsche Forschungsgemeinschaft SFB688 (para A.G.).

Reprodução de rato foi aprovada pelo Regierung Unterfranken, e todas as análises foram realizadas em estrita conformidade com todos os alemão e da União Europeia leis e regulamentos aplicáveis relativos ao uso de animais de laboratório e cuidados.
1. revestimento de microtitulação placas com α4β1 integrina para Ex Utero Allantois explante cultura
<ol><li>Reconstitua a liofilizado murino α4β1 integrina a 200 µ g/mL em salina tampão fosfato (PBS). Dilua para uma concentração final de 10 µ g/mL em pré-aquecido (37 ° C) PBS. Pipete 20 µ l/poço da solução diluída α4β1 integrina para o número necessário de poços de uma placa de microtitulação (1 alantoide/poço). Nota: Evite a formação de bolhas de ar quando o revestimento dos poços da placa de microtitulação.</li>
	<li>Incube as placas de microtitulação para 60 min a 37 ° C, colocando-os em uma incubadora de cultura de tecidos umidificado.</li>
	<li>Aspire cuidadosamente o sobrenadante com uma pipeta. Usar sucção suave e inclinar a placa para evitar tocar bem fundo com a ponta da pipeta. Lavar os poços duas vezes pela adição de 50 µ l de meio de cultura previamente aquecido (37 ° C) como de Dulbecco modificado águia médio (DMEM) e remover o meio de lavagem por sucção suave.</li>
	<li>Passo fundamental: bloco de ligação grátis sites pela adição de 50 µ l/poço de 0,1% (p/v) albumina de soro bovino (BSA) / DMEM e incube por ≥ 30 min a 37 ° C, colocando as placas de microtitulação numa incubadora umidificado cultura de tecidos. Nota: Explantes alantoide podem anexar várias superfícies na presença de soro. Portanto, é imperativo para bloquear sites de ligação não-específica, após o revestimento dos poços com integrina e antes de adicionar o alantoide dissecado por explantes cultura no soro contendo médio (consulte a etapa 4.4).</li>
	<li>Manter os Poços revestidos na solução de bloqueio a 37 ° C, até que seja necessário na etapa 4.4.</li>
</ol>2. o útero dissecação de um rato grávida na Dpc 8
Nota: Reduza taxas de falso-positivo de gravidez, de discriminação de ganho de peso15.
<ol><li>Sacrifica o mouse por deslocamento cervical. Desinfecte o casaco na região abdominal por pulverização com etanol a 70% (v/v). Nota: Utilize luvas quando manusear os ratos. Eutanásia por deslocamento cervical sem anestésicos impede a contaminação química do alantoide, que pode interferir com o acessório de alantoide.</li>
	<li>Abra a pele abdominal, fazendo uma pequena incisão na linha com uma tesoura. Use os dedos com luvas para manter a pele acima e abaixo da incisão e separar a pele em direção ao peito e a cauda do rato.</li>
	<li>Agarre o peritônio com fórceps. Com a outra mão, faça uma incisão em forma de V de anterior para posterior usando a tesoura e mover o tecido para expor na cavidade abdominal. Use a pinça para empurrar os intestinos de um lado para obter acesso ao útero.</li>
	<li>Localize os dois cornos uterinos. Segure o colo do útero (o segmento caudal) com fórceps e corte os ligamentos com uma tesoura bem. Então retire ambos os cornos uterinos por separando os ovidutos com uma tesoura.</li>
	<li>Usar fórceps para transferir o útero em um prato de 10cm estéril contendo estéril, temperatura (RT) PBS. Remova o tecido adiposo, vasos e nervos que cercam os cornos uterinos com pinça fina e/ou tesoura (figura 1A).</li>
</ol>3. embrião dissecação
Nota: Execute as seguintes etapas sob um microscópio estereoscópico equipado com uma gama de zoom de 8:1.
<ol><li>Separe os embriões por corte entre os locais de implantação com uma tesoura. Segure o revestimento do útero muscular dos brotos útero com pinça e puxá-lo distante para expor a decídua (figura 1B).</li>
	<li>Usando fórceps, transferi os gomos do útero para uma nova placa de 10 cm contendo estéril, RT PBS.</li>
	<li>Corrigi a decidua em torno do embrião com as pontas da pinça.</li>
	<li>Fazer uma incisão no lado anti-mesometrial da decidua com pinça fina (Figura 1) e descascar a decidua com fórceps para expor o embrião (Figura 1). Nota: O polo mesometrial da decidua é altamente vascularizado e pode ser mais largo na base que a parte de anti-mesometrial, que contém o embrião e aparece mais pálida.</li>
	<li>Disse o embrião com pinça (Figura 1E, F).</li>
	<li>Usando uma microcolher, transferi o embrião para uma gota de PBS estéril contido em um prato de estéril de 3,5 cm.</li>
	<li>Transferir o embrião para uma gota fresca de PBS no mesmo prato 3,5 cm usando uma microcolher e dissecar o embrião do saco vitelino usando fórceps (Figura 1, H).</li>
	<li>Opcionalmente, se necessário (por exemplo, para a genotipagem por reação em cadeia da polimerase), transferir o saco vitelino em um tubo estéril aspirando com ~ 5 µ l PBS em uma ponta da pipeta orifício grande 200 µ l. Loja a-20 ° C. Nota: Tecido materno (ou seja, o saco vitelino parietal, incluindo a membrana do Reichert e o cone de ectoplacental) que possam contaminar a preparação do saco vitelino pode levar a resultados errôneos de genotipagem. Remoção desses tecidos é mais fácil antes a dissecação do embrião do saco vitelino.</li>
</ol>4. o alantoide dissecção e cultura Ex Utero no imobilizado α4β1 integrina
<ol><li>Corte o alantoide do seu local de inserção basal na extremidade posterior do embrião usando pinça fina (Figura 1 H).</li>
	<li>Opcionalmente, se a plataforma do desenvolvimento é necessária, conta os pares somite do embrião por inspeção visual, usando um microscópio equipado com ótica de contraste de fase e um 5 × 10 × objetivo. Nota: O embrião pode ser estendido com fórceps para facilitar a contagem do somite. Fusão e apego corioalantoicas ocorrem entre E 8.0 e 9.0 E em embriões com pares de 6 somite ≥. Giro começa em embriões com 6-8 pares de somite.</li>
	<li>Pipeta de 40 µ l/poço de DMEM suplementado com 10% (v/v) de soro fetal bovino, L-glutamina e antibióticos no número necessário de poços de placa do microtiter pré-revestida (ver seção 1).</li>
	<li>Recolher o alantoide flutuante aspirando com ~ 5 µ l PBS, em um grande orifício de uma ponta de pipeta de 200 µ l. Transferi 1 alantoide/bem.</li>
	<li>Incubar as allantoides colocando as placas de microtitulação numa incubadora de cultura de tecidos umidificado (37 ° C, 5% CO2) para , por exemplo, 6, 12 e 24 h. Para evitar interferência com o acessório de alantoide, não mova as placas de microtitulação entre esses pontos de tempo.</li>
	<li>Em cada ponto do tempo, marcar acessório alantoide (por exemplo, nenhum acessório/flutuante alantoide alantoide parcialmente ou totalmente anexada; totalmente propagação alantoide) usando um microscópio equipado com ótica de contraste contraste ou diferencial do interferência fase e um × 5 ou 10 × objectivo (Figura 2). Nota: Exemplos representativos de allantoides recém isoladas e totalmente anexadas são mostrados na Figura 2. Ex utero, ponta distal do allantoides isolados de camundongos C57BL/6J de sua anexa dentro de 4-6 h (isto é referido como penhora parcial da alantoide), e acessório completo do alantoide inteira é alcançado dentro de ~ 12 h de cultura. Por 18-24 h, o explante tem aplainado e assumiu uma forma circular (referida para como totalmente espalhar alantoide) com um CD31 positivo do plexo vascular9,16.</li>
</ol>5. confirmação dos coloração o alantoide explantado para o CD31 marcador de células endoteliais
<ol><li>Aspire o meio de cultura de explantes de propagação totalmente e corrigir as células adicionando 40 µ l/poço de 4% (p/v) paraformaldeído/PBS. Nota: Paraformaldehyde é tóxico. Use luvas e óculos de segurança para evitar o contato de olho e pele.</li>
	<li>Incubar durante 10 minutos a RT e, em seguida, aspirar o sobrenadante. Lavar três vezes com a adição de 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave.</li>
	<li>Permeabilize as células e bloquear sites inespecificas adicionando 40 µ l/poço de 0,1% (v/v) de soro de cabra normal BSA/10% Triton X-100/0.1% (w/v) (v/v) em PBS por 30 min em RT. Wash duas vezes adicionando 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave. Nota: O protocolo pode ser pausado a noite neste ponto.</li>
	<li>Mancha de células, adicionando 20 µ l/poço de anticorpos anti-CD31 primário diluído 01:50 em 0,1% (p/v) BSA/PBS durante a noite a 4 ° C. Lavar três vezes com a adição de 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave.</li>
	<li>Detecta anticorpos primários ligados pela adição de 20 µ l/poço de cabra secundário fluorescente etiquetado anticorpos anti-rato diluído 1: 200 em 0,1% (p/v) BSA/PBS por 1h em placas de microtitulação RT. Protect da luz por envolvimento em papel de alumínio ou colocando em uma câmara escura. Lavar três vezes com a adição de 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave.</li>
	<li>Inserir células, adicionando 20 µ l/poço de meio de montagem. Encube por ~ 1h no RT até o meio de montagem tem solidificado. Armazenar placas de microtitulação no RT ou a 4 ° C, protegido da luz.</li>
	<li>Imagem do plexo vascular do alantoide explants sob um microscópio de fluorescência, equipado com um × 5 ou 10 × objetivo e filtros de fluorescência apropriado. Nota: Imagens representativas do plexo vascular CD31 positivo de uma totalmente propagação alantoide são mostradas na Figura 3. Outros marcadores para o plexo vascular, tais como anticorpos dirigidos contra Flk-1, Flt, gravata-1 ou Tie-2 pode ser usado como bem16.</li>
</ol>

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Os autores não têm nada para divulgar.

Na presença de soro, que é uma fonte rica de moléculas pro-adesivo da matriz extracelular como a fibronectina, explantes alantoide prontamente irão anexar vários plástico, vidro ou superfícies de filtro9,16. Assim, se o objectivo do ensaio realizado é especificamente investigar o efeito de uma modificação genética ou qualquer outro tratamento no acessório alantoide para imobilizado α4β1, é fundamental para bloquear sites de ligação não-específi...

A placenta é indispensável para o crescimento embrionário e desenvolvimento no ambiente uterino. Constitui a interface para o oxigênio, metabólito e troca de nutrientes entre a circulação fetal e materna e também funções como órgão endócrino e imunológico. Qualquer insulto endógeno ou exógeno que prejudica o desenvolvimento da placenta pode levar a retardo de crescimento intra-uterino, perda fetal ou complicações na gravidez, tanto em ratos e em seres humanos1. Enquanto o número de mutações específicas do mouse que causam fisiologia placentária anormal continua a aumentar2, com 219 genótipos atualmente listados no site da informática do genoma do rato (MGI) (http://www.informatics.jax.org/vocab/ mp_ontology / MP:0010038), muitos destes fenótipos não são bem compreendidos, do ponto de vista mecanicista. Além de alterações genéticas, drogas pequena molécula, anticorpos monoclonais, toxinas, agentes patogénicos, metabolitos em excesso ou vários agentes ambientais podem afetar desenvolvimento de placenta e função3,4,5 , 6 , 7. ainda, simples em vitro ensaios que passos críticos do modelo em desenvolvimento de placenta são escassos.
Desenvolvimento placentário murino é iniciado no início midgestation, quando o alantoide (o precursor do desenvolvimento do cordão umbilical) emerge da extremidade posterior do embrião como um broto que cresce em direção do córion8. Chorioadhesive células na camada exterior do bud alantoico mediam o apego e a difusão do alantoide na superfície do cório (corioalantoicas "fusão"). O córion dobra-se posteriormente em vilosidades no qual vasculatura do alantoide cresce para formar o labirinto, a camada interna da placenta onde nutrientes e oxigênio são trocados entre o sangue materno intimamente justapostos sinusoides e vasos embrionários9 ,10.
O anexo do alantoide para córion é o passo inicial e fundamental na formação do labirinto, e defeitos neste processo estão entre as causas mais comuns de letalidade embrionária em midgestation1. Embora um número de mutantes de rato tem sido descrito onde corioalantoicas ceda a ocorrer10, e o banco de dados do MGI atualmente lista 108 mutantes de rato que caracterizam-se por fusão corioalantoicas anormal (http:// www.Informatics.Jax.org/Vocab/mp_ontology/MP:0002824), a interação intercelular entre as células vasculares adesão molécula-1 (VCAM-1, expressado na mesoderme alantoico) e α4β1 integrina (expressado no mesotélio gonadotrofina coriônica, que é derivado do mesoderma extraembryonic) parece ser indispensável para a ligação corioalantoicas e fusão11,12,13. Coloração imuno-histoquímica de todo-monte selvagem-tipo embriões tem mostrado que o VCAM-1 é expresso dentro os dois terços distais do caule alantoico no dia aproximadamente embrionário (E) 7,513 (palco do somite 1-4), e que sua expressão permanece elevada durante corioalantoicas anexo e - fusão11,12. Falha de alantoico apego ao córion normalmente é detectada por análise histológica de botões do útero. No entanto, alantoide tamanhos são fisiologicamente altamente variáveis entre e mesmo dentro de ninhadas do mesmo grau de desenvolvimento14, e o alantoide só pode anexar o córion quando atingiu um tamanho suficiente para fazer contato físico. Reflectindo esta variabilidade natural, anexo corioalantoicas ocorre em algum momento entre ~ E 8.0 E 9.0 e no uteroe uma avaliação estatisticamente confiável deste processo pela histologia depende, portanto, a análise de um grande número de conceptuses para obter suficiente amostras na fase do desenvolvimento apropriado.
Aqui, descrevemos um método para avaliar fixação alantoico ex utero que é menos dependente do tamanho do alantoide. Demonstramos a dissecação de embriões do rato e seus allantoides desde o útero de ratos grávidas ~ 8 dias post coitum (dpc; dpc 0.5 designa a detecção de um tampão vaginal), e a cultura subsequente do alantoide explants na α4β1 imobilizado integrinas. Este método permite uma avaliação rápida, funcional de acessório e espalhando o comportamento de vários explantes alantoide em paralelo e em pontos diferentes de tempo sucessivos. O protocolo pode ser usado para tela para efeitos de diversos fatores ambientais, drogas ou mutações específicas no alantoide acessório ex vivo.

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			<td height="23" style="height:23px;width:319px;">C57BL/6J wildtype mice</td>
			<td style="width:256px;">The Jackson Laboratory</td>
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			<td height="21" style="height:21px;">70 % (v/v) Ethanol</td>
			<td style="width:256px;">VWR International</td>
			<td>930031006</td>
			<td></td>
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			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>11000-12</td>
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			<td height="21" style="height:21px;width:319px;">Micro dissecting forceps</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>Dumont # 5 medical biology forcepts with fine, sharp tip.&#160;</td>
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			<td height="21" style="height:21px;width:319px;">Fine scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>14094-11</td>
			<td>Straight blades.</td>
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			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td style="width:256px;">Fine Science Tools</td>
			<td>15012-12</td>
			<td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microspoon</td>
			<td style="width:256px;">Carl Roth</td>
			<td>AT18.1</td>
			<td>5 mm Spoon diameter.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Microtiter plates</td>
			<td style="width:256px;">ibidi</td>
			<td style="width:119px;">81501</td>
			<td>We use uncoated, sterile angiogenesis slides (internal volume 50 &#181;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-a4b1-mediated binding to plastic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">10 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150350</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">3.5 cm dish</td>
			<td style="width:256px;">Nunc</td>
			<td style="width:119px;">150288</td>
			<td>Sterile tissue culture dishes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large orifice pipette tips&#160;</td>
			<td style="width:256px;">Biozym</td>
			<td style="width:119px;">VT0140X</td>
			<td>Low binding pipette tips, 200 &#181;L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a4b1 integrin</td>
			<td style="width:256px;">R&#38;D Systems</td>
			<td style="width:119px;">6054-A4</td>
			<td>Murine a4b1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:319px;">Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>Without Ca2+ and Mg2+.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:319px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-03600</td>
			<td style="width:915px;">The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO3 but no L-glutamine, which is added separately.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:319px;">Penicillin/Streptomycin</td>
			<td style="width:256px;">Gibco (ThermoFisher Scientific)</td>
			<td style="width:119px;">15140-122</td>
			<td>100 x (10,000 U/mL Penicillin and 10,000 &#181;g/mL streptomycin) stock solution.&#160;Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">L-Glutamine</td>
			<td style="width:256px;">PAN Biotech</td>
			<td style="width:119px;">P04-80100</td>
			<td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &#176;C to avoid freeze/thaw.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Fetal bovine serum&#160;</td>
			<td style="width:256px;">Biochrom</td>
			<td style="width:119px;">S 0115</td>
			<td>We use heat-inactivated FBS (heated to 56 &#176;C for 30 min with mixing to inactivate complement).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin (BSA)</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">A7030</td>
			<td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &#181;m pore size and low protein-binding membrane, and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">para-Formaldehyde</td>
			<td style="width:256px;">Carl Roth</td>
			<td>0335.2</td>
			<td style="width:915px;">To make a formaldehyde solution in PBS, weigh para-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &#176;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &#176;C for approximately four weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Triton X-100</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td style="width:119px;">X100</td>
			<td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Normal goat serum</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>G9023</td>
			<td>To block non-specific binding of antibodies in immunofluorescence analyses.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">a-CD31 Antibodies</td>
			<td style="width:256px;">BD Biosciences</td>
			<td>550274</td>
			<td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary goat anti-rat antibodies</td>
			<td style="width:256px;">Thermo Fisher</td>
			<td>A-11006</td>
			<td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mowiol 4-88</td>
			<td style="width:256px;">Sigma Aldrich</td>
			<td>81381</td>
			<td>Mounting medium for cytochemistry. MW ~31,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Labovert&#160;</td>
			<td style="width:256px;">Leitz</td>
			<td style="width:119px;">Labovert&#160;</td>
			<td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">M80</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">M80</td>
			<td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">DM4000B</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">DM4000B</td>
			<td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Digital camera</td>
			<td style="width:256px;">JVC</td>
			<td style="width:119px;">KY-F75U</td>
			<td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:319px;">Confocal microscope</td>
			<td style="width:256px;">Leica Microsystems</td>
			<td style="width:119px;">SP5</td>
			<td style="width:915px;">Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td>
		</tr>
	</tbody>
</table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

A placenta é essencial para o crescimento e o desenvolvimento de embriões de mamíferos. Por este motivo, inúmeras alterações genéticas e insultos ambientais também prováveis que perturbam o desenvolvimento da placenta ou função podem causar perda de gravidez precoce em ratos e seres humanos. No entanto, simples em vitro ensaios para a tela para potenciais efeitos sobre a formação da placenta são escassos. Aqui, focalizamos o primeiro e fundamental passo na formação da placenta, que consiste de penhora do alantoide com o cório de modelagem. Nós descrevemos um método para avaliar rapidamente o acessório de explantes alantoico na imobilizado α4β1 integrina, que serve como substrato cório-mimética. Esta abordagem in vitro permite uma avaliação qualitativa do acessório e espalhando o comportamento de vários explantes alantoide em pontos diferentes vez consecutiva. O protocolo pode ser usado para investigar o efeito de mutações específicas do mouse, drogas ou vários fatores ambientais que têm sido associados a complicações na gravidez ou perda fetal no alantoide acessório ex vivo.

Este protocolo descreve um método para isolar e explantes murino allantoides ex vivoe detalha a avaliação qualitativa do alantoide penhora a α4β1 integrina, um processo crítico durante na vivo corioalantoicas fusão. Os representante resultados mostrados na figura 1A–H demonstram as etapas sucessivas de isolamento alantoide a partir do útero gestante. Tecidos maternos que cercam os cornos uterinos, ...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Descrevemos um ensaio em vitro de fixação corioalantoicas de modelo, o primeiro passo na formação da placenta. O protocolo demonstra a cultura de dissecção e explante de murino allantoides em imobilizado α4β1 integrina. Acessório de alantoide é avaliado microscopicamente em pontos de tempo pré-determinado.

Dissecção e cultura de explante de murino alantoide para a análise In Vitro de fixação alantoico

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

dissection-explant-culture-murine-allantois-for-vitro-analysis

Research

JoVE Journal

Medicine

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

7.8K visualizações.  University of Würzburg. Descrevemos um ensaio em vitro de fixação corioalantoicas de modelo, o primeiro passo na formação da placenta. O protocolo demonstra a cultura de dissecção e explante de murino allantoides em imobilizado α4β1 integrina. Acessório de alantoide é avaliado microscopicamente em pontos de tempo pré-determinado.

Vídeo: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

Dissecção da Human Elements Corpo Vítreo para Análise Proteômica

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Medicina

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Pele da biópsia Explant Cultura para Derivação de primárias fibroblastos humanos

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusões artéria carótida para farmacocinéticas e farmacodinâmicas Análise de taxanos em Ratos

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

Whole humor vítreo Dissecção de Análise Vitreodynamic

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Murino próstata Micro-dissecção e castração cirúrgica

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

Análise otimizada de<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; A esteatose hepática

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Dissecação do pâncreas Mouse para análise histológica e perfis metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

Técnica de Explante Coclear Murino Neonatal como Ferramenta de Triagem In Vitro na Pesquisa Auditiva

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Análise quantitativa de Micro-CT de Aortopathy em um modelo do rato de β-aminopropionitrile-induzida de aneurisma da aorta e dissecação

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...